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H-NS controls metabolism and stress tolerance in Escherichia coli O157:H7 that influence mouse passage
Irfan Erol1,
Kwang-Cheol Jeong2,
David J Baumler3,
Boris Vykhodets2,
Sang Ho Choi4 &
Charles W Kaspar2,3
H-NS is a DNA-binding protein with central roles in gene regulation and nucleoid structuring in Escherichia coli. There are over 60 genes that are influenced by H-NS many of which are involved in metabolism. To determine the significance of H-NS-regulated genes in metabolism and stress tolerance, an hns mutant of E. coli O157:H7 was generated (hns::nptI, FRIK47001P) and its growth, metabolism, and gastrointestinal passage compared to the parent strain (43895) and strain FRIK47001P harboring pSC0061 which contains a functional hns and 90-bp upstream of the open-reading frame.
The hns mutant grew slower and was non-motile in comparison to the parent strain. Carbon and nitrogen metabolism was significantly altered in the hns mutant, which was incapable of utilizing 42 carbon, and 19 nitrogen sources that the parent strain metabolized. Among the non-metabolized substrates were several amino acids, organic acids, and key metabolic intermediates (i.e., pyruvate) that limit carbon acquisition and energy generation. Growth studies determined that the parent strain grew in LB containing 14 to 15% bile or bile salts, while the hns mutant grew in 6.5 and 9% of these compounds, respectively. Conversely, log-phase cells of the hns mutant were significantly (p < 0.05) more acid tolerant than the parent strain and hns mutant complemented with pSC0061. In mouse passage studies, the parent strain was recovered at a higher frequency (p < 0.01) than the hns mutant regardless of whether log- or stationary-phase phase cells were orally administered.
These results demonstrate that H-NS is a powerful regulator of carbon and nitrogen metabolism as well as tolerance to bile salts. It is likely that the metabolic impairments and/or the reduced bile tolerance of the E. coli O157:H7 hns mutant decreased its ability to survive passage through mice. Collectively, these results expand the influence of H-NS on carbon and nitrogen metabolism and highlight its role in the ability of O157:H7 strains to respond to changing nutrients and conditions encountered in the environment and its hosts.
Escherichia coli O157:H7 is a human pathogen that causes hemorrhagic colitis in humans and in some cases may incite hemolytic uremic syndrome [1]. Cattle are a principal reservoir and contaminated ground beef is most frequently implicated in foodborne outbreaks associated with this human pathogen [2, 3]. Adult cattle harboring serotype O157:H7 strains are asymptomatic, and the numbers and duration of shedding are influenced by a number of factors including feed, the microbial composition of the intestinal tract, the time of year, and environmental sources [4]. The complex interplay between the bovine host and E. coli O157:H7 persistence has not been fully elucidated although Naylor et al. [5] recently demonstrated localization and adherence to mucosal epithelium in the recto-anal junction. The changing conditions encountered by E. coli O157:H7 in the bovine intestinal tract and the environment require that it closely control metabolism and stress protection.
H-NS is a cytoplasmic protein that is abundant in the E. coli nucleoid [6]. It plays a role in the condensation of chromosomal DNA by binding to curved DNA and consequently can influence the expression of many genes either directly or indirectly [7, 8]. The level of H-NS in cells remains relatively constant throughout log- and stationary-phases although hns transcription is subject to auto repression [9, 10]. The production of over 60 proteins in E. coli are either transcriptionally or translationally controlled by H-NS [8, 9, 11]. Several genes modulated by H-NS are responsive to changes in environmental conditions like pH, osmolarity, and temperature [8, 9, 11–15]. The involvement of H-NS in modulating genes responsive to environmental stimuli make it an important regulator of virulence in several pathogens including Shigella and Salmonella [12, 13, 16–19].
Although there has been considerable work on gene regulation by H-NS, there has been comparatively little work on its impact on bacterial metabolism and stress tolerance. Considering the varied and changing environments encountered by E. coli O157:H7, a detailed understanding of the consequences of modulators that are responsive to environmental conditions is particularly important. To this end, an hns mutant was constructed to define the growth and metabolic changes and any subsequent impact on gastrointestinal passage in this important human pathogen.
Generation and confirmation of the hns::nptI mutant
A 695-bp fragment that includes the hns structural gene and upstream regulatory region was amplified by PCR from genomic DNA of E. coli O157:H7 ATCC 43895. The nucleotide sequence of the Eco RI-Bam HI fragment was determined and was identical to the sequenced gene in E. coli O157:H7 EDL933 [20]. Transconjugants resulting from the conjugation of pSC0063 from E. coli SM10 λpir to E. coli O157:H7 strain ATCC 43895 were kanamycin resistant, ampicillin sensitive, sucrose positive, and O157 positive. Confirmation of a double crossover in which wild-type hns was replaced with hns::nptI was confirmed by PCR (data not shown). PCR analysis of genomic DNA from the parental strain (ATCC 43895) with primers choi9917 and choi9918 generated a 375-bp fragment; whereas, genomic DNA from the hns::nptI mutant (FRIK47001P) resulted in a DNA fragment approximately 1.6-kb in length. The 1.6-kb fragment is in agreement with the projected size of the DNA fragment containing wild-type hns (375 bp) and the nptI gene (1.2 kb).
Growth and serological characteristics
The growth of the hns mutant (FRIK47001P) in LB at 37°C was slower and reached a lower maximum optical density than the parent strain or FRIK47001P containing pSC0061 (data not shown). The doubling times of the parent, complemented, and mutant strains were 27, 34, and 36 minutes, respectively. The maximum OD600 nm achieved by the parent and complemented strains was 1.3 to 1.4 after 18 h of incubation while the hns mutant reached a maximum OD600 nm = 1.0. Similar to the results in LB, the growth of the hns mutant in LB supplemented with mucin or in fecal slurries was slower and reached a lower final density than the parent strain. In addition, the colony size of the hns mutant on agar media was smaller than the parent and complemented strains. The presence of kanamycin in the growth media did not influence the growth of the mutant. These results are in agreement with results with hns mutants of non-pathogenic E. coli which had greater doubling times than the parent strains [11, 21]. One contributor to the longer doubling time is the increased σs level in the hns mutant, which is partially suppressed in hns rpoS double mutants [11]. The hns mutant tested negative for the H7 antigen and was non-motile in motility medium (data not shown); however, the O157 antigen was still detected. The absence of H7 is consistent with previous data showing that H-NS is involved in the control of flagella gene expression and that hns mutants are non-motile [22, 23].
Growth inhibition by pH, NaCl, and bile
In addition to slower growth, the hns mutant was more susceptible to inhibition by low pH, NaCl, bile (Table 1) and cefixime (data not shown) compared to the parent and complemented strains. Low temperature further slows the growth of hns mutants [24]. Thus, the growth of the mutant was further restricted when low temperature was combined with another inhibitor. The minimum growth pH of the hns mutant at the different temperatures was 4.5 to 5.0 which was 0.5 to 1.0 pH unit higher than the parent and complemented strains with the greatest difference observed at the lowest incubation temperature (16.5°C). Both the parent and complemented strains grew at pH 4.0 at each incubation temperature. Similar to the results with pH, the hns mutant grew in LB supplemented with a maximum of 4–6% NaCl compared to 6–7% for the parent and complemented strains. The test strains tolerated the highest concentration of NaCl when incubated at 25°C. There was one exception in which the hns mutant complemented with pSC0061 grew in a maximum of 4% NaCl at 16.5°C which was equivalent to the maximum level tolerated by the hns mutant. It is unclear why the complemented strain differed from the parent strain in this case where in all other growth conditions the results were the same. An unexpected finding was the ability of the E. coli O157:H7 to grow in LB containing bile compounds at 14–15%. The hns mutant was significantly more sensitive to bile salts and bovine bile preparations and grew in the presence of a maximum of 6.5% and 9.0% of these compounds, respectively. The growth of the strains in the presence of bile was further studied in LB containing 5% bile salts. Although the hns mutant grew, it had a greatly extended lag period (approximately 40 h) in comparison to the parent and complemented strains (<12 h) (Figure 1).
Table 1 Growth of Escherichia coli O157:H7 strains in LB as influenced by pH, temperature, NaCl, and bile compounds.
Effect of bile salts on the growth of E. coli O157:H7 43895 (parent) and hns mutant (FRIK47001P). Growth of the parent strain (○), hns mutant (□), and hns mutant complemented containing pSC0061 (Δ) in LB containing 5% bile salts number three at 37°C. Data points are the average values from three trials.
Acid tolerance
The acid tolerance of the parent, hns mutant, and complemented strains was tested using both log- and stationary-phase cells. The survival of log-phase cells of the hns mutant was significantly greater (p < 0.05) than the parent strain when challenged in synthetic gastric fluid (pH 2.0) (Figure 2A). These findings are consistent with the derepression of σs (rpoS)-regulated genes in the absence of H-NS. Stationary phase increased the acid tolerance compared to log phase in all three strains and the numbers decreased by less than 1 log10 CFU/ml after 3 h of acid challenge (Figure 2B). However, the parent and hns-complemented strains exhibited similar acid tolerance that was significantly greater (p < 0.05) than the hns mutant.
Acid tolerance of E. coli O157:H7 43895 (parent) and hns mutant (FRIK47001P). Survival of the parent strain (●), hns mutant (■), and hns mutant complemented containing pSC0061 (▲) in simulated gastric fluid (pH 1.5) at 37°C when using log-phase (A) and stationary-phase (B) cells. Data points are the average values from at least three trials. Error bars represent the standard error of the mean.
In contrast to the findings with acid challenges conducted in synthetic gastric fluid that is acidified with hydrochloric acid, there was no difference in the survival of the parent and hns mutant strains challenged in LB acidified with acetic or lactic acids (data not shown).
Glutamate decarboxylase activity
In an attempt to discern the reduced acid tolerance in stationary-phase cells of the hns mutant, glutamate decarboxylase (GAD) activity was measured (ΔpH/h/108 CFU). The hns mutant had significantly less (p < 0.01) GAD activity than the parent strain and strain FRIK47001P complemented with pSC0061 (p < 0.001) when examining stationary-phase cells (data not shown). These findings provide one possible explanation for the reduced acid tolerance in comparison to the parent strain of the hns mutant in stationary phase.
Sole carbon and nitrogen sources
Metabolic characterization using Phenotype Microarrays showed that the hns mutant was incapable of utilizing 42 carbon and 19 nitrogen sources that the parent strain metabolized (Tables 2 and 3). The ability of the hns mutant to uptake and/or use a variety of substrates as sole carbon sources was significantly altered. The parent strain was able to use six amino acids as sole carbon sources that the hns mutant was unable to utilize. The mutant did not use several disaccharides (rhamnose, lactulose, sucrose, maltose) and trehalose, dulcitol, D-ribose, and dextrin. In addition, eight organic acids, including pyruvate, were not used as a sole carbon sources by the hns mutant (Table 3). H-NS is involved in the regulation of genes associated with carbohydrate metabolism, like the maltose operon [25], but there were no carbon sources that were metabolized by the mutant that were not utilized by the parent strain. The parent strain metabolized six amino acids as sole nitrogen sources that the hns mutant was unable to use; however, the mutant was capable of using several dipeptides (Table 3). Nitrite was one nitrogen source that was utilized by the hns mutant but not the parent strain (data not shown). The findings from this metabolic characterization greatly expand the role of H-NS as a regulator of carbon and nitrogen metabolism in E. coli.
Table 2 Sole carbon source metabolism by the E. coli O157:H7 parent (ATCC 43895) and hns mutant (FRIK 47001) strains.
Table 3 Sole nitrogen source metabolism by the E. coli O157:H7 parent (ATCC 43895) and hns mutant (FRIK 47001) strains.
Gastrointestinal passage
Testing of fecal samples prior to inoculation determined that mice were E. coli O157:H7 negative and that few bacteria grew on the selective agar that were similar to the inoculation strain (i.e., sorbitol negative). The administration of the parent strain, hns mutant (FRIK47001P), and hns mutant complemented with pSC0061 did not result in signs of illness in ICR mice. Although mice were tested for 3 days post inoculation, all mice that shed the inoculated E. coli O157:H7 strain tested positive one day after inoculation and some mice continued to shed the strain at day 2. No fecal samples tested positive at day 3. The results from three trials are shown in Table 4. The parent strain was recovered from a total of 13 mice of 27 inoculated with log-phase cells (3 trials and 3 inoculation doses) while only a single mouse receiving the hns mutant tested positive at a dose of 103 CFU. The results between the parent and hns mutant were statistically different (p < 0.01) at a dose of 104. Similar results were obtained with stationary-phase cells of the parent strain, which was detected in a total of 17 of 27 mice inoculated. The passage of the hns mutant was not improved when stationary-phase cells were administered, and the feces from only 1 mouse (dose of 104 CFU) of 27 inoculated tested positive. At doses of 103 and 104 CFU, the results with stationary-phase cells of the parent strain were statistically different (p < 0.05 and p < 0.01, respectively) from those obtained with the hns mutant. The passage of the hns mutant complemented with a functional hns in pSC0061 was similar to that of the parent strain (data not shown). The diminished passage of the hns mutant was further demonstrated when the percent of the dose surviving passage through the mouse was calculated. Approximately, 7.9% of the parent strain and 4.2% of the hns mutant complemented with pSC0061 were recovered in feces compared to only 0.2% of the hns mutant that was determined from the few fecal samples that harbored the mutant. These results demonstrate that H-NS significantly impacts gastrointestinal passage in mice.
Table 4 Recovery of E. coli O157:H7 strains ATCC43895 (parent) and FRIK47001P (hns mutant) from feces following oral administration to ICR mice.a
E. coli O157:H7 must respond to changing environmental conditions whether it is present in the gastrointestinal tract or the environment [26]. Metabolic pathways must be carefully controlled and stress-protection systems triggered in a timely manner to effectively compete and survive. H-NS is a major component of the bacterial nucleoid, having pleiotropic effects on gene expression, genome stability and DNA recombination [9, 27]. H-NS is involved in the control of several genes in E. coli associated with the metabolism of specific substrates as well as stress protection [8, 9, 15]; such as Lrp (leucine-responsive regulatory protein), σs (rpoS), and two-component systems (PhoP, EvgA and YedW) [8]. Thus, H-NS is a master regulator affecting the expression of individual genes and regulons with several of the phenotypes observed in hns mutants occurring as a result of indirect effects on gene expression [8, 27].
In our studies of an hns mutant of E. coli O157:H7, the growth was slower than the parent strain. Although it is not completely understood, the increased doubling time in hns mutants is partially related to the increased level of σs and the possibility of secondary site mutations that affect growth [11]. H-NS negatively influences the level of σs at the posttranscriptional level [9, 28, 29], and the level of σs increases in log-phase cells of hns mutants [11, 30]. Another factor that contributes to the level of σs in hns mutants is the increase in the heat-shock chaperone DnaK which enhances σs stability [15]. The increase in σs results in the production of σs-regulated proteins some of which are involved in stress protection [8, 15, 30]. Results from this study using phenotypic microarrays showed that H-NS has a significant impact on carbon and nitrogen metabolism that likely impact the growth of the mutant (Tables 3 and 4). Among the substrates that were not metabolized were several carbon and nitrogen sources, including several key metabolic intermediates (Figure 3) that likely influence growth and energy generation. It is probable that the slower growth of the hns mutant is a result of the combined effects of the increased level of σs [11] and altered substrate metabolism. However, another possibility that cannot be excluded is the generation of a second site mutation that influenced growth [11, 40].
Key carbon sources that were not metabolized by the E. coli O157:H7 hns mutant. Some of the metabolic intermediates and substrates that were not (□, open box) metabolized as sole carbon sources by the hns mutant of E. coli O157:H7 when examined using Phenotypic Microarrays. Both the parent and hns mutant strains metabolized the compounds that are not in boxes. Compounds in italics which were not tested (a). E. coli converts glutamate and oxaloacetate to α-ketoglutarate and aspartate (b) [55,56]. Glycyl-L-proline was tested (c). α-ketoglutarate was not metabolized as a sole carbon source by neither the parent strain nor the hns mutant (d).
The growth of the hns mutant was also more susceptible to inhibition by NaCl and bile salts than the parent strain (Table 1). These differences were accentuated by sub-optimal growth temperature, often encountered when outside of the intestinal tract. A significant finding from these growth comparisons was the high tolerance of the parent E. coli O157:H7 strain to bile and bile salts (15 and 14%, respectively), while the growth of the hns mutant was limited to a maximum of 9.0 and 6.5%, respectively. Bile salts are produced by the liver to breakdown fat and are deleterious to biological membranes. E. coli must tolerate bile salts that can reach concentrations of 20 mM in the duodenum and even higher in the small intestine [31]. The reduced capacity of the mutant to tolerate bile salts might be a consequence of membrane changes in the hns mutant. H-NS is known to influence expression of at least 5% of the genes in E. coli, many that encode for membrane proteins [8]. The possibility of an altered membrane is supported by the inhibition of the mutant by the cephalosporin-like antibiotic, cefixime, while the parent strain grew in media containing 0.4 μg cefixime/ml. Future studies with H-NS-deficient strains should be useful in identifying the proteins involved in bile tolerance in E. coli O157:H7 strains.
Acid tolerance is one characteristic that influences the persistence of E. coli O157:H7 shedding in cattle [32] and may promote survival during gastric passage in humans [33]. The increased acid tolerance of the hns mutant in comparison to the parent strain in log-phase cells is consistent with the expression of σs-regulated proteins [9]. In stationary-phase cells, the acid tolerance increased in all three strains (parent, hns mutant, and hns complemented strains) in comparison to log-phase cells. However, the hns mutant was less acid tolerant than the parent strain in stationary phase. The gad system comprised of GadA and GadB, two isoforms of glutamate decarboxylase, and gadC that encodes for a glutamate/γ-aminobutyrate antiporter [34–38] is one contributor to acid tolerance. Results from the phenotype microarrays showed that the hns mutant did not utilize glutamate as a sole carbon source; therefore, we compared glutamate decarboxylase activity in stationary-phase cells of the mutant and parent strains. The specific activity of glutamate decarboxylase was reduced in the hns mutant (p < 0.01) compared to the parent strain. The decreased specific acitivity of glutamate decarboxylase may explain the difference in acid tolerance between the hns mutant and parent strain in stationary-phase cells.
In mouse passage studies, recovery of the hns mutant was significantly less than the parent strain (Table 2). The mutant was recovered from a single mouse in each of three trials conducted with log-phase and stationary-phase cells. It is possible that the reduced ability of the hns mutant to survive mouse passage is a consequence of a depleted energy supply, resulting from its limited capacity to metabolize available substrates, which is necessary for the optimal functioning of homeostasis or stress protection systems (i.e., ATPase). Gene expression profiles of mucus-grown E. coli have identified genes involved in the catabolism of gluconate, N-acetylglucosamine, sialic acid, glucuronate, mannose, fucose, and ribose are induced and considered preferred carbon sources [39]. Mutants incapable of metabolizing these seven carbohydrates have a decreased capacity to colonize streptomycin-treated mice. Although the mouse passage assay employed in this study did not evaluate colonization, the results from Phenotype Microarrays found that five of the seven carbohydrates identified by Chang et al. [39] as preferred carbon sources were utilized by the hns mutant while the E. coli O157:H7 parent strain used all seven carbon sources (see Tables 2 and 3). In addition, the hns mutant grew in mucin-supplemented LB broth and fecal slurries to a concentration essentially equivalent to that of the parent strain although the lag-phase was longer and the growth rate slower than that of the parent strain which is similar to the results obtained in growth studies conducted in LB. Therefore, a defect in the ability to utilize a mucin carbohydrate is probably not the reason for the reduced passage of the hns mutant.
There are a number of possible antimicrobial factors in the gastrointestinal tract that may be responsible for the diminished capacity of the hns mutant to survive passage through ICR mice. Acetate can reach a concentration of 70 mM in the intestinal tract [41, 42]. Although log-phase cells of the mutant were more acid tolerant in challenges conducted with hydrochloric acid, additional challenges were conducted with acetic and lactic acids because results from phenotype microarrays found that the mutant did not utilize acetate or lactate as sole carbon sources. It was reasoned that tolerance to these acids might be impaired, but acid challenges with lactic acid and acetic acid at pH 4.4 found no difference in the survival of the parent and hns mutant strains with log- or stationary-phase cells (data not shown). Another possibility is that bile salts may have affected passage of the hns mutant through mice. In addition to being inhibitory, bile in association with H-NS is known to repress virulence genes [43]. However, additional experiments are needed with bile-sensitive mutants and measurements of bile acid concentrations in the mouse intestinal tract to confirm that bile was responsible for the reduced recoveries of the hns mutant. Considering that the intestinal tract contains at least 500 bacterial species [39], it is also possible that one of the commensal microbes produces an antimicrobial factor that is detrimental to the hns mutant. Studies with gnotoxenic mice would be particularly valuable in addressing the contributions of competing flora on the survival of the hns mutant. Based upon the results from this study, the most plausible explanation for the reduced passage of the hns mutant through mice is the regulatory impairments that influence growth and stress protection.
Results from this study significantly expand the importance of H-NS in carbon and nitrogen metabolism (Figure 3) as well as stress tolerance that collectively influence the ability of E. coli O157:H7 to respond to changing conditions it encounters in its host and the environment. This is the first report of the high tolerance of E. coli O157:H7 strains to bile and bile salts that may be of significance in localization and persistence within its bovine host and possibly during human infection. Studies are in progress to identify, which proteins that are influenced by H-NS contribute to bile tolerance. The results from this study also establish H-NS as a potential target for control of this important human pathogen.
Bacterial strains and culture conditions
The bacterial strains and plasmids used in this study are listed in Table 5. E. coli were grown in Luria-Bertani (LB) broth at 37°C. Antibiotics (Sigma Chemical Company, St. Louis, MO) were added to media when appropriate: ampicillin, 100 μg/ml; kanamycin, 50 μg/ml; and tetracycline, 10 μg/ml. For log-phase and stationary-phase cultures, 5 μl of an overnight culture was used to inoculate 5 ml of LB and incubated with shaking (150 rpm) at 37°C to an optical density at 600 nm = 0.5 (log phase, ~108 CFU/ml) and optical density at 600 nm = 1.1 (stationary phase, ~109 CFU/ml), respectively.
Table 5 Plasmids and E. coli strains used in this study.
Generation and confirmation of an hns::nptI mutant
A DNA fragment containing the hns structural gene and flanking regions was amplified from genomic DNA of E. coli O157:H7 ATCC 43895 by PCR using oligonucleotide primers (choi0005, 5'-TTTGGATCCAAAGCCTGGCTTGAAGAAGAGATG-3' and choi0006, 5'-TTTGAATTCTTTTGAATTCCTTACATTCCTGGC-3') containing an Eco RI (choi0005) and Bam HI (choi0006) site on the respective 5'-ends. After PCR amplification, the PCR fragment was digested with Eco RI and Bam H1 and ligated into pBR322, previously digested with the same enzymes, to produce pSC0061. The nucleotide sequence of the cloned fragment was determined (University of Wisconsin-Biotechnology Center). The suicide vector, pCVD442, was used to generate hns mutant in E. coli O157:H7 ATCC 43895 by homologous recombination. The hns gene in pSC0061 was inactivated by insertion of nptI encoding for aminoglycoside 3'-phosphotransferase (confers resistance to kanamycin) digested from pUC4K [44] using Pst I and gap filled with Klenow fragment. npt I was ligated with a partial Pst I digest of pSC0061 carrying hns which has a Pst I site present within its open reading frame (ORF). The construct (pSC0062) was digested with Eco R1 and Bam H1 to liberate the 1.9-kb hns::nptI fragment. The fragment was blunt-ended with Klenow and ligated with Sma I-digested pCVD442 [45] to produce pSC0063. The constructed vector was conjugally mobilized from E. coli SM10 λ pir [46] carrying the tra gene to recipient strain E. coli O157:H7 43895. Conjugation and mutant selection were conducted as described previously [47]. Transconjugants that were kanamycin resistant, ampicillin sensitive and tested positive for the O157 antigen by latex agglutination (Oxoid, Basingstoke, England) were selected for further study. The primers choi9917 (5'-ATGAGCGAAGCACTTAAAATTCTGAAC-3') and choi9918 (5'-TTCTTCCATTGCTTTTTTGATTACAGC-3') were used to amplify a portion of the hns ORF and confirm the presence of nptI in hns (1.6-kb fragment) (data not shown). FRIK 47001 (hns::npt I) was complemented by transforming FRIK 47001 with pSC0061.
The analysis of sole carbon and nitrogen source utilization was conducted with Phenotype MicroArrays™ (Biolog, Hayward, Calif.). This method is used to identify the function and global interactions of a gene on cellular metabolism [48, 49]. The parent E. coli O157:H7 strain ATCC 43895 and hns::npt I mutant (FRIK47001) were evaluated for their ability to use carbon and nitrogen sources using PM1, PM2, and a PM3 plates, respectively. Strains were grown on R2A agar (Biolog) overnight at 37°C and sufficient cells were transferred to inoculating fluid to equal the 85%T turbidity standard (Biolog). FRIK47001 was resuspended in inoculating fluid containing glucose rather than succinic acid when inoculating the PM3 plate because it was unable to use succinic acid as a carbon source. Each well was inoculated with 100 μl of the standardized cell suspension, incubated at 37°C for 48 h, and the results recorded.
Growth studies
Growth studies were conducted in LB adjusted to the appropriate pH or supplemented with NaCl, bile, bile salts, or cefixime using the parent, hns mutant, and hns mutant complemented with pSC0061. Cells were used to inoculate the adjusted LB medium at a final concentration of 105 CFU/ml and incubated at 16.5, 25, and/or 37°C without shaking. The minimum pH for growth was determined in the pH range of 3.5 to 7.0 at 0.5 increments. The following were added to LB at the following ranges: NaCl, 0 to 10% (0.5% increments); bile salts no.3 (Difco), 0 to 16%; bovine bile (Sigma), 0 to 16%; porcine stomach mucin (Sigma), 2.5 mg/ml; bovine submaxillary gland mucin (Sigma), 2.5 mg/ml; and cefixime (Lederle), 0.2 to 0.8 μg/ml. Inoculated tubes were incubated at the designated temperature and observed daily for turbidity for up to 21 days. The growth of the strains in the presence of bile salts and mucin were studied in more detail. Growth in LB supplemented with 5% bile salts or 2.5 mg mucin/ml was monitored spectrophotometrically at 600 nm using a Bioscreen Analyzer at 37°C for 48 h (Labsystems, Helsinki, Finland). Plates were shaken for 30s prior to each optical density reading. The growth of strains was also monitored in fecal slurries (mouse feces diluted 1:10 in sterile deionized water). The fecal slurries were inoculated at a final concentration of approximately 105 CFU/ml from overnight cultures grown in LB, and then incubated at 37°C. Samples were removed and the number of CFU/ml determined by plating on MacConkey sorbitol agar (MSA, Difco) supplemented with 2.5 μg potassium tellurite/ml (Fisher) and 0.05 μg cefixime/ml (Lederle Laboratories, Pearl River, NY) [50] and enumerating the number of sorbitol-negative colonies.
Acid challenge
Acid tolerance was assessed in synthetic gastric fluid adjusted to pH 1.5 and LB acidified with acetate or lactate (pH 4.4) as previously described [51]. Log (OD600 = 0.5) and stationary (OD600 = 1.1) phase cells were used to inoculate flasks containing 50 ml of synthetic gastric fluid or acidified LB to achieve a final concentration of ca. 105 CFU/ml. Flasks were incubated at 37°C with shaking (150 rpm). Samples were removed periodically and then plated on tryptic soy agar (TSA, Difco) using a Model D Spiral Plater (Spiral Systems Inc., Cincinnati, OH). The number of colony forming units (CFU) was determined after 24 h of incubation at 37°C. The survival curves shown are the mean values from at least three trials.
GAD activity
The cells were examined for GAD activity as previously described [52, 53] with the following modifications. Strains were cultured overnight in LB at 37°C for 21 h. The cells from 1.0 ml were pelleted by centrifugation and washed with 0.9% NaCl. The cells were resuspended in 3.0 ml of GAD reagent (1 g of L-glutamic acid, 0.05 g of bromocresol green, 90 g of NaCl, and 3 ml of Triton X-100 per liter) and incubated for 3 h at 35°C. The color and pH change of the reaction mixture was recorded every 30 minutes. GAD activity was reported as the ΔpH/h/108 cells. The mean values were calculated from at least three trials.
Mouse inoculation studies
The passage studies were conducted as previously described [32]. In brief, strains were grown in LB at 37°C to log and stationary phase. After harvesting and washing with PBS, the cells were resuspended in 10% sucrose or phosphate buffer (0.01 M, pH 7.4) to a concentration of 104, 103, or 102 CFU/ml and administrated to individually housed ICR mice (ca. 20 g, Harlan Sprague Dawley Inc., Madison, Wis). Three trials were conducted, and in each trial three mice were inoculated with each concentration of the test strain. Fecal samples were collected each day for 3 days after inoculation and tested for the presence or absence of E. coli O157:H7. Fecal samples were diluted (1:10) in modified EC broth supplemented with novobiocin (20 μg/ml) and incubated at 37°C with shaking (150 rpm) for 18–24 h. Following enrichment, 20 μl of Dynabeads® anti-E. coli O157 (Dynal Biotech ASA, Oslo, Norway) was added to 0.5 ml of the enriched fecal culture and wash buffer (0.5 ml), incubated, and washed as described by the manufacturer. The resuspended beads were streaked on MacConkey sorbitol agar supplemented with potassium tellurite/ml and cefixime/ml [50]. The plates were incubated at 42°C for 24 h and examined for sorbitol-negative E. coli O157:H7 colonies. Suspect colonies were confirmed as O157 by agglutination (E. coli O157:H7 test kit, Oxoid). The minimum detection level of this procedure was approximately 100 CFU/g.
The data presented are the mean values from at least three trials. The acid tolerance of strains was compared using the inverse of slopes from linear regression plots of survivor curves. The data from acid challenges were analyzed for statistical differences by one-way analysis of variance. A z-test for was used to evaluate significant differences in the proportions of mice testing positive between inoculation strains [54]. All calculations were conducted using SigmaStat software (Jandel Scientific, San Rafael, Calif.).
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We wish to thank Jeff Bose, Clayton Hiemke, Billy Hung, and Barbara Cochrane for technical assistance and valuable discussions. The work was supported in part by a grant from the USDA, NRICGP awarded to CWK and the College of Agricultural and Life Sciences, University of Wisconsin-Madison.
Department of Food Hygiene and Technology, University of Ankara, Ankara, Turkey
Irfan Erol
Department of Food Microbiology and Toxicology, University of Wisconsin, Madison, Wisconsin, 53706-1187, USA
Kwang-Cheol Jeong
, Boris Vykhodets
& Charles W Kaspar
Cellular and Molecular Biology, University of Wisconsin, Madison, Wisconsin, 53706-1187, USA
David J Baumler
Department of Food Science and Technology, Seoul National University, Seoul, 151-742, South Korea
Sang Ho Choi
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Correspondence to Charles W Kaspar.
IE, KCJ, DJB, and BV conducted laboratory experiments, analyzed data, and contributed to the writing of the manuscript. SHC contributed to the initial concept of this project and constructed and confirmed the hns mutant and pSC0061. CWK designed and coordinated the project and wrote the manuscript.
Parent Strain
Glutamate Decarboxylase

redpajama_set_name: RedPajamaCommonCrawl


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short snippets: LADIES IN WAITING.
New Chookies ready to be glazed and fired again.
Some new designs also need to be glazed with a clear glaze.
Well this time I have been making lots of new designs, which was a lot of fun, but it took me longer to do of course, used different clays as well.
And by doing that I have to do different firings because these clays need different temperatures.
A few domino's for the culture to culture challenge.
Another kiln load for the first firing and then I can finally finish al these pieces.
Very exiting new designs! nice nice!


TACOMA, WASHINGTON – Paulo Sergio Alfaro-Sanchez, an illegal immigrant being held at a detention center in Washington state, had no idea that the federal government would count him in the census.
But counted he was, along with other illegal immigrants facing deportation in detention centers across the country – about 30,000 people on any given day, according to U.S. Immigration and Customs and Enforcement.
This census brings a twist, though. For the first time, states have the option of counting people in detention centers and prisons as residents of their last address before they're detained, worrying some local lawmakers who say cities and counties that host detention centers could lose money.
"Detention centers and prisons should probably count where they are located, that's where resources would be required," Rep. Sanford D. Bishop, D-Georgia wrote in a May letter to the chairman of the subcommittee that oversees the census. Bishop represents Stewart County, Georgia, population 4,600, where the nation's largest detention center housed a total of 14,000 people between April 2007 and March 2008.
ICE operates 22 immigrant detention centers and also houses people in hundreds of other jails or prisons. Most of the largest centers are in small towns in Texas, Arizona and Georgia. Texas is home to six detention centers, and Arizona has three.
The payout can be hefty for small towns. Federal money being distributed from the census averaged about $1,469 per person in fiscal year 2008, according to the Brookings Institution, and other grants are also available to small towns depending on their population.
In Raymondville, Texas, a town of nearly 10,000 people, the Willacy Detention Center holds an average daily population of about 1,000. The center opened in 2006 and was a boon to the community as ICE and the private company that runs the center rushed to hire personnel.
Now, the detention center's population may push Raymondville over the town's goal of surpassing 10,000, a number that will allow them to qualify for more federal help, Mayor Orlando Correa said.
"As long it's humane, as long as the facility respects the rights of these people and they're not treated like animals, I'm OK with it," Correa said.
For safety reasons, most detainees are counted through administrative records, rather than forms being passed out, U.S Census Bureau spokesman Stephen Buckner said. The census will cull data from records kept on April 1.
Alfaro-Sanchez, for his part, is glad he's being counted. He entered the country when he was about 15 through Tijuana, and worked as a handyman in Goldendale, a small town in eastern Washington.
He arrived in at the Northwest Detention Center in Tacoma on March 30 after being arrested in a fight. The charges were dropped, he said, but immigration officers had already flagged him for arrest.
"I think that even though we may be sent back, there's a lot of people who may need that money, the Hispanic people that are here," the 32-year-old said in Spanish.
GAINESVILLE, FLORIA – When Kelly Stauff saw the man in the crosswalk, it was too late to stop. She didn't hit the man, who turned out to be a Gainesville police officer in street clothes, but Stauff was nailed with a $154 ticket for failing to yield to a pedestrian.
Stauff and others have complained about GPD's tactics in its new initiative to improve pedestrian safety, saying they are sneaky and create a violation that wouldn't have existed otherwise.
GPD counters that such sting details are endorsed by pedestrian and bicycle organizations, and are an effective way to get across a message that traffic laws will be enforced.
And it's likely that GPD may do more of them when schools reopen after the summer break, said Cpl. Tscharna Senn.
Senn added that an early morning accident downtown on May 22 in which three pedestrians were hit is a prime example of why both pedestrians and drivers need to be more alert when crossing streets.
Senn added that officers won't determine who was at fault in that accident until an analysis of evidence is done.
GPD has already issued far more tickets for failing to yield to pedestrians in crosswalks this year than in past years: 9,174 so far compared with 9,043 last year and 10,026 in 2008.
OREGON – Almost two years after a 12-year-old Calgary boy was whisked into foster care in the United States in a bizarre custody dispute, an Oregon court judge has decided he's coming home.
Noah Kirkman will be back home in Canada in a few weeks, but there was no suggestion that anything was amiss that caused the youngster to be kept from his family for almost two years.
"Noah was happy," said Tony Merchant, the Regina-based lawyer for the family of the boy, who will finish his school year in Oregon.
Mr. Merchant, who has been involved in the case for the last few months, said that something was truly "bizarre" in the handling of this case. But he didn't blame the judge who led it.
Noah also met with his grandparents Thursday night, looked at his Calgary home on Google Earth and is excited to be reunited with his parents and sister, Mr. Merchant said.
The court acknowledged that there are still transition issues, but Oregon officials have been told to work them out.
The boy, who had been caught in bureaucratic limbo since the summer of 2008, will be returning to his Canadian family in a few weeks, the judge ruled.
The legal nightmare began when Noah was vacationing with his stepfather in small-town Oregon, while his mother and younger sister remained at home in Canada.
The boy was riding his bike without a helmet when he was stopped by police, but had trouble answering questions. He has severe attention deficit hyperactivity disorder, but maintains an A average in school. Officials checked out his background and found an open social services file in Canada, which was the result of his special needs assistance, and that he was in the U.S. without his mother, his legal guardian and deemed her note permitting care by his stepfather wasn't enough.
Noah was taken into custody to protect his welfare, although Oregon's Department of Human Services won't talk about the case citing privacy rules.
Noah's mother, Lisa, and stepfather, John, who now resides with the family in Calgary and is the father of Noah's sister Mia, (he and his wife for a time lived in different cities) have been fighting to be reunited with Noah ever since.
Last month, Oregon's Lane County Circuit Court Judge Kip Leonard ruled that he might be open to sending the boy back to Canada when the school year ends, but there was no guarantee.
SAN BERNARDINO, CALIFORNIA – A father is behind bars, arrested for allegedly kidnapped his own children from California 14 years ago, and bringing them to Central Florida to live.
For years, investigators have been searching for him, but it was the social networking website Facebook that delivered the break it took more than a decade to get, MyFoxOrlando reports.
Faustino Utrera is now charged with two counts of kidnapping, and two counts of violating child custody orders.
According to the Osceola County Sheriff's Office, Utrera was at a bus stop on Wednesday at about 2:30 in the afternoon, waiting to pick up his 16-year-old son from school, when he was taken into custody.
Faustino's 16-year-old son and 17-year-old daughter had been attending celebration high school. Investigators said the family of three had actually been living in the palm key mobile home park in Polk County.
Investigators said that in 1995, Utrera took his two kids from the family's San Bernardino, California home and disappeared.
Then just last march, investigators said the mother of the children was on Facebook and found her daughter. When she began conversing online, her very own daughter, who hadn't seen her since she was 3 years old, told her mom she wanted nothing to do with her and deleted her Facebook page.
San Bernardino authorities issued an out-of-state arrest warrant, and this past Wednesday, Osceola County deputies caught up with Utrera.
We know Faustino's daughter is about to turn 18, and she officially graduated from celebration high school on Thursday night. For now, both children are in the custody of the State of Florida.
NEW JERSEY – A Buffalo man accused of running a multibillion-dollar online gambling site for a New York City crime family posted $100,000 bail this week following a court appearance on money-laundering and illegal gambling charges.
Investigators with New Jersey's Division of Criminal Justice accuse Brian R. Cohen, 62, of operating a "wire room" in Costa Rica, where the Lucchese crime family took in $2.2 billion in illegal bets on sporting events.
Cohen has residences in Buffalo and Costa Rica.
He was arrested May 16 on a return trip from Costa Rica at Buffalo Niagara International Airport by detectives from the New Jersey Division of Criminal Justice Gangs & Organized Crime Bureau.
Cohen allegedly ran the Lucchese family's offshore gambling operations, including a Web site called http://www.bigactionsports.com. The wire room in San Jose, Costa Rica, had up to 20 people at a time taking bets by phone and processing wagers, according to Stephen J. Taylor, director of the Division of Criminal Justice.
Cohen appeared Thursday in New Jersey Superior Court in Morristown, where Judge Thomas V. Manahan ordered him to surrender his U. S. passport.
His arrest was "another significant milestone" in the prosecution of the Lucchese crime family, Taylor said.
"We have indicted the top echelon of this criminal organization in both New York and New Jersey, and we continue to charge key players in their multibillion- dollar gambling operation," he said.
A New Jersey grand jury earlier returned indictments against 34 alleged Lucchese members and associates, including two of the family's "ruling members," on multiple counts including racketeering, conspiracy and money laundering.
The allegations against Cohen also will be presented to a grand jury.
If convicted, Cohen could face up to 10 years in state prison on the money-laundering charge and five years on the gambling charge.
WEST YORKSHIRE, UK – Marcus Morris was told that police they had taken his VW Polo because the open window – which he had accidentally left down – meant the vehicle was at risk of theft.
Mr Morris, 25, had parked the car near to Leeds city centre as he went for a job interview but returned an hour-and-a-half later to find it had gone.
He contacted West Yorkshire Police believing it had been stolen but was told they had removed it to protect it from thieves.
Mr Morris, said: "I couldn't believe it. It seems outrageous.
Police told him a window had been left open and a CD wallet was in the back of the car. The vehicle had been taken to a storage facility.
When Mr Morris collected his car the next morning he was handed a bill for £150. "I'm not working at the moment and that's a lot of money for me. It's money I don't think I should have had to pay," he said.
West Yorkshire Police said it made no apology for taking action. Chief Insp Elizabeth Belton said: "Officers regularly patrol our communities looking for opportunities to prevent crime, which is exactly what has happened on this occasion.
"The vehicle was left insecure with valuables on show in an area with significant levels of car crime and we were unable to locate the owner.
"We make absolutely no apology whatsoever for the officer's actions, which have prevented this vehicle becoming a target for thieves. Had a thief got there first it would have been a very different story.
The charges made for the recovery and storage of vehicles are set nationally by the Government. Mr Morris confirmed he is taking legal advice in an attempt to recover the cost.
PHOENIX, ARIZONA – Maricopa County sheriff's officials said Tuesday that financial sanctions imposed by the Board of Supervisors will affect public safety, and the office likely will challenge the moves in court.
Sheriff's Office attorneys have an 8:30 a.m. telephone conference today with a Superior Court judge on the proposed actions.
The conflict between the two entities hit a fever pitch Monday when the board froze the Sheriff's Office's access to racketeering and jail-enhancement funds starting July 1 because of what it suspects may be misspending. The funds totaled about $3 million this year. The move came after Sheriff Joe Arpaio refused to hand over financial documents the board subpoenaed. The board is trying to investigate the spending and set Arpaio's budget for next fiscal year.
The board is scheduled to consider a range of additional financial actions against the sheriff at 11:30 a.m. today. The potential sanctions include canceling Sheriff's Office credit cards, closing its outside bank accounts, restricting non-emergency travel or putting the office's $269 million budget on line-item. The board had promised the actions would not impact public safety.
Still, Sheriff's Office spokeswoman Lisa Allen said any actions would have serious ramifications and could affect everything from street patrols to drug investigations and disaster response. For example, she said, the Sheriff's Office will have to park about 100 patrol cars funded exclusively through the racketeering funds if the money cannot be spent.
And if the board canceled staff credit cards, Allen said, deputies would be unable to quickly gas up patrol cars, purchase airline tickets for 20 to 30 monthly extraditions or purchase supplies to keep the office running.
"If we don't have the flexibility to react as law enforcement is made to react – which is quickly – because they're a little ticked off at us with how we're reacting to their request, that will hinder our ability to keep the public safe," Allen said.
Sheriff's Office attorneys and staff also said the board could only put the office on a line-item budget if it was over budget. Sheriff's spokesman Brian Lee said the office was within its $269 million budget and had spent about $215 million through April.
Allen said attorneys almost certainly would request a temporary restraining order to block any sanctions.
County spokeswoman Cari Gerchick said the county has an obligation to the public to take action.
The board since March has asked the sheriff to turn over financial documents including detailed credit-card transactions, work assignments, a list of all detainees or defendants extradited, and a list of all bank accounts dating to 2005.
The board subpoenaed the information and gave Arpaio until May 7 to turn over the documents. When he missed the deadline, the board tried to convene a contempt hearing. Attorneys for the Sheriff's Office appealed to the court.
Pima County Superior Court Judge Richard Gordon ruled the board could not force Arpaio to attend the hearing and scheduled a hearing for June 4.
Sheriff's attorney Kerry Martin said today's proposed actions by the board could violate the temporary restraining order.
He said the action is "direct retaliation" for not turning over the documents. Martin requested a telephone hearing with Gordon to ask the judge to consider another temporary restraining order against the board if it imposes sanctions.
The board wants the records to help set Arpaio's budget but also believes he may be misspending or misusing public funds, though to what extent is uncertain.
For years, county government was conducted without much fanfare.
Budgets were negotiated, misspending dealt with quietly. Audits were low key. The supervisors tussled with a few department heads and elected officials over money and power, but those cases didn't match today's outright war with Arpaio.
Betsey Bayless was a supervisor in the 1990s, when the board and sheriff first fought over authority and money.
The supervisors and their attorneys maintain that if there is misspending, it must be rooted out. They also point out the new fiscal year begins soon.
Critics have said the county's executive branch has used its budget authority to go after enemies.
"The board and county management use their powers of the purse to amass other powers and to settle grudges with other elected county officials," said former County Attorney Andrew Thomas, who resigned to run for attorney general.
Last summer, seven countywide elected officials met to talk about a perceived power grab and lack of respect by the board and top county administrators.
But today's relationships with the board are strong, said three elected officials, because supervisors Max Wilson and Don Stapley have reached out to meet regularly.
County Recorder Helen Purcell, Assessor Keith Russell and Schools Superintendent Don Covey all said they have positive relationships with the board and the Office of Management and Budget.
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Q: Taking Screenshots using Selenium Web Driver How to take Screenshots using Selenium Web Driver ?
I am using a Grid Configuration having 2 nodes. I need to take screen shots at every defined interval from both nodes.
Please do not specify the way to take screenshot. I want the Test cases not to call a common method every time to take screenshot. Rather another thread should be always active which can take screenshots at an interval.

A: You will have to have multiple threads, and pass the driver instances to both of them, and one thread will perform the test, while another thread will sit on a timer, taking a screenshot every X seconds.
I don't recommend this, however, here's why:

*

*It's probably going to throw a lot errors.  What happens if you try to take a screenshot while the driver is booting up, or going to a page?  It may not even have the capability to take a screenshot while doing simple things.

*Unless you are going to be taking screenshots every half second, and putting it into a video, (which is going to slow down your test), the majority of the screenshots are not going to be useful, and the useful ones are going to be hard to find in the massive amount of screenshots taken.

*If you want to include another driver, you're going to have to pass it to the thread as well, taking a third screenshot.
So, in conclusion, I ask why you want this...because I would be happy to suggest an alternative solution that will still fit your needs.

redpajama_set_name: RedPajamaStackExchange


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Dozens of non-urgent surgeries postponed due to flu at St. Boniface
Published Monday, January 13, 2020 6:10PM CST
(File image)
WINNIPEG -- With high numbers of people headed to Winnipeg hospitals with the flu, St. Boniface Hospital is making room by postponing or rescheduling dozens of elective or non-urgent surgeries this week.
A Winnipeg Regional Health Authority spokesperson tells CTV News that from Monday to Wednesday 28 procedures have been postponed and another 7 have been rescheduled to happen at either Pan Am Clinic or Victoria Hospital.
A press release from St. Boniface Hospital explained that postponing surgeries opens up temporary beds for patients who are in immediate need of care during flu season.
The hospital also issued the following statement:
"It is important to restate that urgent and emergent surgeries continue to take place. This includes the prioritization of elective cancer surgeries and cardiac surgeries. Some elective surgeries may be rescheduled at a later date or occurring at other health care facilities. Patients whose surgeries are affected will be contacted by their surgeon's office 2 to 3 days ahead of their surgery date if their surgery is rescheduled. Elective surgery beds are being re-designated for patients with influenza on an as-needed basis. This means scheduling adjustments are made day-to-day," told CTV News in an email.
The WRHA has also transferred patients to at least one hospital outside the city. The spokesperson said to date fewer than five people have been moved from Winnipeg to Brandon for care and that those patients could be transferred safely.
Flu activity in Manitoba is still considered to be high according to the latest weekly provincial flu update. Both Influenza A and B, as well as a high volume of patients with other respiratory illnesses, appear to be surging at the same time this year.
The WRHA says it's ICU is still operating at overcapacity.
Health Authority officials also thank staff for their work during this busy time.


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Home > News > Bookselling
This Week's Bestsellers: March 11, 2019
By Carolyn Juris |
Strong Border
Don Winslow's The Border, which our starred review called the "stunning conclusion to his monumental Cartel trilogy," enjoyed a nice bit of real-world symmetry, pubbing just weeks after the conviction of El Chapo, the inspiration behind one of the series' main characters. It debuts at #3 in hardcover fiction; on March 7, FX announced a deal to adapt the series for TV.
(See all of this week's bestselling books.)
New Yorker staff writer Patrick Radden Keefe hits #8 in hardcover nonfiction with Say Nothing, which our starred review called "a moving, accessible account of the violence that has afflicted Northern Ireland." Keefe's investigation of the 1972 disappearance and murder of a Belfast mother of 10, he told PW in November, had an unexpected result: "What I did not realize when I embarked on this project is that I would end up discovering the identity of the individual who actually pulled that trigger in 1972—and identifying that person, for the first time, in the book."
Tui T. Sutherland's middle grade fantasy series Wings of Fire has sold more than two million print copies over seven years and 12 novels. In 2018, a graphic edition of the first book, The Dragonet Prophecy, illustrated by Mike Holmes, pubbed to modest first-week print unit sales. It's gone on to sell 92K print units, and now the second graphic adaptation, The Lost Heir, pubs with first-week print unit sales of 15K. The book lands at #2 on our children's fiction list.
The 7 ½ Deaths of Evelyn Hardcastle
Stuart Turton
#17 Hardcover Fiction
Turton's debut, which our review said "blends mystery with Groundhog Day and Quantum Leap," lands on our list for the first time with its best weekly sales since its September publication.
#3 Trade Paperback
The author of The Alice Network returns with another historical thriller, set in the aftermath of WWII. Our review said it "vividly reveals how people face adversity and sacrifice while chasing justice and retribution."
California Girls
Susan Mallery
"Mallery fans and newcomers alike will adore this tale," our review said, "about the bonds of sisterhood and friendship tested by life's ups and downs."
1 Green Eggs and Ham Dr. Seuss Random House 43,795
2 Becoming Michelle Obama Crown 41,690
3 One Fish Two Fish Red Fish Blue Fish Dr. Seuss Random House 35,464
4 The Cat in the Hat Dr. Seuss Random House 30,019
5 Where the Crawdads Sing Delia Owens Putnam 27,074
6 Brawl of the Wild (Dog Man #6) Dav Pilkey Graphix 26,134
7 Girl, Wash Your Face Rachel Hollis Nelson 22,740
8 Fox in Socks Dr. Seuss Random House 21,589
9 The Threat Andrew G. McCabe St. Martin's 21,569
10 There's No Place Like Space Tish Rabe Random House 20,283
All unit sales per NPD BookScan except where noted.
A version of this article appeared in the 03/11/2019 issue of Publishers Weekly under the headline: Behind the Bestsellers
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When you create a new project, you'll be directed to the project creation form. This page is not accessible to the public. Only you alone can consult it when connected to your Ulule account.
You can take as long as you like to write the description of your project, add images or videos, make changes, organise your rewards.
The moderation team will not review your project until you click "Submit my project."


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Schedule of Classes - September 9, 2021 7:14PM EDT
Course Catalog - September 9, 2021 7:15PM EDT
Introduction to Computer Game Design
A project-based course in which programmers and designers collaborate to make a computer game. This course investigates the theory and practice of developing computer games from a blend of technical, aesthetic, and cultural perspectives. Technical aspects of game architecture include software engineering, artificial intelligence, game physics, computer graphics, and networking. Aesthetic and cultural aspects include art and modeling, sound and music, game balance, and player experience.
Prerequisites/Corequisites Prerequisite for programmers: CS 2110 or permission of instructor. Prerequisite for designers: INFO 2450 and/or submission of art/design samples to instructor. Corequisite: ENGRC 3152.
Distribution Category (SMR-AS)
Satisfies Requirement Satisfies track requirement for INFO majors and minors if taken under the INFO prefix.
Regular Academic Session. Choose one lecture and one discussion. Combined with: CS 3152
10871 INFO 3152 LEC 001
MWF 10:10am - 11:00am Online Meeting
Feb 8 - May 14, 2021
White, W
Instruction Mode: Online
Admittance to CS/INFO 3152 is by instructor permission only. Students who would like to obtain permission must submit an application. The application is available online at http://gdiac.cs.cornell.edu/courses/application
Instructor Consent Required (Add)
10872 INFO 3152 DIS 201
TR 11:20am - 12:10pm Online Meeting
TR 12:25pm - 1:15pm Online Meeting
Disabled for this roster.


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RLC's The Magnolia Residences to receive award
May 7, 2019 | 12:02 am
ROBINSONS Land Corporation (RLC) said its project The Magnolia Residences in Quezon City will receive an award at the Asia Pacific Property Awards (APPA) in Bangkok this May.
The four-tower residential project, which is part of the five-hectare Magnolia Town Center, is recognized as award winner for the Philippines. It will also compete against other Asia-Pacific development in the Best Residential High-Rise category.
The Magnolia Town Center was previously named by APPA as the Best Mixed-Use Development in the Philippines 2013-2014.
"The Asia Pacific Property Award recognition is a testament to RLC's unwavering commitment to continuously craft high-quality developments that truly add value to Filipino lives," said Henry L. Yap, senior vice president and business unit general manager of RLC Residential Division.


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Home >> January 2018 Edition >> An XTAR Focus
An XTAR Focus
Combating interference
Team Leader: "Mission Control, this is Team Leader. In position. Waiting video feed. Over."
Mission Control: "Mission Control. Wait. Out."
Team Leader: "Team Leader. We are in position. Is mission on? Window is small, and all elements in position. Just need that drone video. Over."
Mission Control: "Mission Control. Wait. Video corrupt. Attempting to correct. Over."
Team Leader: "Team Leader. What do you mean video corrupt? Is the drone up there? I need that video before final insertion!"
Mission Control: "Mission Control. I think we have some interference going on. Seems to be messing up the video feed. Over."
Team Leader: "Team Leader. Window closing. Targets moving out. I must have that video now, if we are to continue the mission. Over."
Mission Control: "Mission Control. Something's definitely interfering with the video. There's nothing I can do. Advise mission abort. Over."
Team Leader: "Team Leader. Mission abort!? Are you crazy? We've been planning this for weeks. We'll have to go in without the intel. Over."
Mission Control: "Mission Control. Team Leader, video is definitely being interfered with. Mission is compromised. Abort. Move to extract point and return to base. Out."
Much focus has been devoted to satellite interference recently, as well it should be. Interference is a serious issue for satellite operators and the problem will likely worsen.
Artistic rendition of XTAR-EUR X-band services satellite. Image is courtesy of XTAR.
The increase in number of satellites orbiting, the rise of High Throughput Satellites (HTS) employing multi-spot beams and the reduction in antenna size all mean that the potential for interference will only increase.
When discussing satellite interference, the tendency is to categorize it into two main types that users typically encounter; adjacent satellite interference and accidental interference.
When government or military users experience either form of interference, their mission is delayed and in some cases endangered. Time is lost and, in many instances, additional funds must be spent to remedy the problem.
To decrease the chance of any type of satellite interference, government and military users should rely on those frequencies dedicated solely to their use, such as Mil-Ka-band and X-band.
These frequencies have natural advantages that automatically translate into a lower chance of interference and therefore a greater chance to complete the mission.
When a user experiences interference, they are often assigned a new frequency, but it's not always possible to do so if there is no other frequency available.
Finding the source of the interference can often be difficult, and in many cases, is never discovered. Regardless of the type of interference or its cause, it can be a nuisance in the best-case scenario and a disaster in the worst.
Adjacent Satellite Interference (ASI) often occurs when using small terminals, typically sub-1.0 meter, a common size for Comms-On-the-Move (COTM) or Comms-On-the-Pause (COTP) situations.
This is due to beam size; the smaller the terminal the wider the beamwidth. Therefore, small terminals are more prone to transmitting and receiving ASI.
Commercial band satellites, such as Ku- and Ka-band, are typically spaced apart at two degrees (sometimes even closer). This spacing makes users with small terminals vulnerable to ASI, as their beamwidths are larger than two degrees. In example, a parabolic Ku-band antenna smaller than about 0.8 meters would have a 3 dB beamwidth larger than two degrees in the transmit direction.
To avoid transmitting ASI, the EIRP spectral density (a measure of radiated power per unit bandwidth) is strictly limited in Ku- and commercial Ka- frequency bands. To stay within these limits, users must often use a technique called spread spectrum.
Spread spectrum allows a user to transmit the higher EIRP often needed by a small terminal to close a link by spreading the power over a larger bandwidth. However, doing so increases cost.
The result of spreading the signal can double, triple or quadruple the amount of bandwidth required. With this significant increase in bandwidth, the user may experience a cost higher than what might have been expected.
Orbital slots for satellites using military frequencies (X-band and Mil-Ka) are typically spaced four degrees apart (twice the spacing as their commercial counterparts). This wider spacing allows the coordinated EIRP density limits to be significantly higher than those at Ku-band and commercial Ka-band. The higher EIRP density limits allow even smaller sub-meter terminals to transmit, in most cases, without the need to use spread spectrum.
The second type of interference is accidental. The most common types of accidental interference occur when a user accidentally transmits at the incorrect frequency or transmits on the incorrect polarization.
Through the company's experience providing X-band, noted is that there is a significantly smaller base of users, as the frequency is reserved for military and other government users. While a smaller base of users creates a smaller chance of causing accidental interference, it is also true that when interference does occur, there is a smaller base of users to contact to correct the issue.
Cross-pol interference is a specific type of accidental interference and occurs when a terminal transmits at the correct frequency, but on the opposite polarization from that assigned. It is here that military frequency bands have a distinct advantage.
Ku-band and some commercial Ka-band operates with linear polarization and, to avoid interference, users must ensure their feed is properly aligned, either vertically or horizontally. This alignment of the feed on the terminal can be affected by wind or snow from a strong storm.
From the misalignment, that user's signal can begin to bleed into the cross-pol transponder and into another user's signal. In contrast, X-band and Military Ka-band both use circular polarization which does not require alignment. Therefore, users do not have to be concerned about the accidental misalignment of the antenna feed.
A discussion on interference would not be complete without mentioning jamming. Jamming is, of course, purposeful interference. With frequencies that are reserved for government and military use, we see a smaller chance of interference due to jamming.
Commercial Ku- and Ka-band equipment that can be used for jamming is readily available. In contrast, terminals operating at the military frequency bands are less available to those seeking to do harm and are mostly under the control of the military.
This means there is less likelihood that rogue militants would be able to jam communications on a satellite using military frequencies.
As more and more technologies arise, unfortunately the chance for interference also rises. Much discussion and effort have been devoted to mitigating interference. Nowhere is this more important than for the military user.
By using those frequencies specifically dedicated to the military and government user, the chances for interference are naturally reduced.
www.xtar.com
Founded in 2001, XTAR, LLC is the first commercial satellite operator to provide services in the X-band frequency. XTAR is a privately owned company backed by majority shareholder Loral Space & Communications of New York. XTAR also enjoys investment and support from minority shareholder Hisdesat Strategic Services SA.
XTAR launched its fleet without government funding, employing its own technical and financial resources for a system that is reserved exclusively for the benefit of the government user. Countries around the world trust XTAR to support critical services such as border security and information gathering.
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using System;
using System.Globalization;
using System.Windows.Media.Animation;
using Microsoft.VisualStudio.TestTools.UnitTesting;
using SharpTestsEx;
using Topics.Radical.Windows.Presentation.Converters;

namespace Test.Radical.Windows.Presentation.Converters
{
    [TestClass]
    public class TimeSpanToKeyTimeConverterTests
    {
        [TestMethod]
        [TestCategory("Converters")]
        public void Should_be_able_to_convert_a_timespan_into_a_keytime()
        {
            var timespan = TimeSpan.FromSeconds(123);
            var converter = new TimeSpanToKeyTimeConverter();

            var keytime = (KeyTime)converter.Convert(timespan, typeof(KeyTime), null, CultureInfo.InvariantCulture);

            keytime.TimeSpan.Should().Be.EqualTo(timespan);
            keytime.Type.Should().Be.EqualTo(KeyTimeType.TimeSpan);
        }

        [TestMethod]
        [TestCategory("Converters")]
        public void Should_be_able_to_convert_back_a_keytime_into_a_timespan()
        {
            var keytime = KeyTime.FromTimeSpan(TimeSpan.FromSeconds(123));
            var converter = new TimeSpanToKeyTimeConverter();

            var timespan = (TimeSpan)converter.ConvertBack(keytime, typeof(TimeSpan), null, CultureInfo.InvariantCulture);

            timespan.Should().Be.EqualTo(keytime.TimeSpan);
        }
    }
}

redpajama_set_name: RedPajamaGithub


"Given (lit., 'being that it is known") that the reward for good deeds and the good we will merit if we observe G-d's ways as written in the Torah is the World to Come, as it is stated, 'In order that He will do good to you and you will lengthen days' (Deut. 22:7). [Conversely,] the revenge which is exacted from the wicked who have forsaken the paths of righteousness written in the Torah is excision, as it is stated, 'That soul shall be utterly cut off; its sin is in it' (Numbers 15:31).
In the last class we began discussing an important auxiliary issue to the Rambam's discussion. The Rambam here discusses the issue of the temporal reward the Torah promises us for our good deeds. Being that the true reward we will receive is the eternal bliss of the World to Come, what role do these much more temporary blessings fulfill? Will a portion of our reward be granted to us in this world — detracting from the much greater reward in store for us in the next one?
This law of the Rambam is quite long, so I will translate his answer G-d willing next time. Last time, however, we raised an entirely different issue, which too requires serious examination. Our tradition teaches us that the true reward for all our actions will be in the World to Come. As the Talmud puts it, "There is no reward in this world" (Chullin 142a). The physical comforts and pleasures of this world are all fleeting and inconsequential. They are not even "reward" per se. At least according to our religion, they have no part in the ultimate bliss of Heaven.
If so, something is enormously bothersome. Why does the Torah — the Written Torah that is — make virtually no mention of the World to Come? If it's so basic to Judaism, why can't Scripture be more explicit about it? Why is it only discussed in the Oral Law — the part of our tradition handed down orally from Sinai, only much later recorded (in part) in the Mishna and Talmud? The Written Torah seems to promise only reward in this world for our good behavior — timely rains, bountiful harvests, good health, peace and prosperity. How can it possibly leave out something so fundamental to Jewish theology?
We answered this question in part last week, establishing the basic principle. This time we will expand on it and examine a very telling application.
The written and oral parts of the Torah serve entirely different purposes. The Oral Torah contains the bulk of the Torah's wisdom, providing Judaism's philosophical underpinnings and serving as a detailed guide for how to live in this world. The Written Torah is not so much an instruction book, and it is certainly not a philosophical work discussing religion. It is the story of the world.
The Written Torah tells us the story the world — how it was created near perfection, how it fell, and the origins and development of plan to restore it to its previous glory and ultimately bring it to its perfection.
In the Garden of Eden the world was almost perfect. Adam and Eve had only to pass a single challenge and the world would have immediately reached its perfected state. They fell and the world fell with them. In outline form, the Torah continues with the story of Abraham and his descendants — how they discovered G-d once again and began creating a nation which would bring back His message to mankind. They were granted the Torah and brought to the Holy Land — where they were to create a utopian society, in which they would teach man — primarily through example — how the world may once again be brought to perfection.
Based on this, our basic question falls away immediately. The Written Torah is not a work of theology. Its purpose is not to discuss G-d or matters metaphysical. It does not even mention topics so basic as the World to Come, the Resurrection, or man's fate after he dies. It left all of that in the Oral Law's capable hands. Rather, the Written Torah is the story of the physical world.
Further, when we think about it, the Torah's presentation of the mitzvos (commandments) is also entirely different based on this.
The Written Torah, however, does nothing of the sort. It much more prosaically promises rains and good harvests. Why not something loftier? The reason is again because the Torah is the story of this world — and so, it presents the mitzvos as the means through which this world may be improved. It instructs us in mitzvah observance not in order to earn reward in a world after this one, but as a means of bringing this world back to its perfected state. If we obey G-d's will, the physical world will be "fixed"; it will operate in harmony with the spiritual. The rains will come, peace will prevail, disease will not strike. Every aspect of creation will work together. The world fell because of sin; through the mitzvos of the Torah we can fix it anew.
Based on this, we can understand another basic distinction between the Written and Oral Law. The Written Torah discusses the mitzvos only very broadly, leaving the vast majority of the details for the Oral Law. Why is that? Why leave so much out?
The answer is that the Written Torah discusses only general values — the basic types of behavior required to perfect the world. They outline the basic direction the world must take in order to fix its problems. And when we think about it, including too many details would obscure that overall message.
This is a perfect illustration of the devil being in the details. Once we get into the many details of observance, the Torah's overall value system might easily be missed. As a simple example, the Torah teaches us to rest on the Sabbath. Once we get into the nitty-gritty, how one specific act of separating a food from its inedible portions is more "restful" than another similar act this will hardly be evident. It is not, of course, that the details of the Oral Law are less binding or significant, but that they will quickly and of necessity obscure the overall goal and message of the mitzvos — as in the case with any complex legal system. As just and meaningful as the Torah's laws are, on the atomic level the overall message will be difficult to discern. The Written Torah therefore had to de-emphasize the details to accurately convey the big picture.
This topic is very broad and could easily go well beyond the scope of this article. What I'll therefore do is provide one brief but telling example and wrap up.
The answer is that the Written Torah is conveying a message — what G-d's values truly are in such a situation. When you damage your fellow, it is not just the financial loss. Paying him money will never truly make up his loss. The only true compensation is for you to suffer the same loss you inflicted on your fellow. That is the only justice which would "fix up" this world again, returning the world to its pristine state prior to your sin.
Next time, G-d willing, we will finally get to the Rambam's answer to his question.


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Taforalt, une ville du Maroc.
 L' Homme de Taforalt ou Ibéromaurusien, un type anthropologique.

redpajama_set_name: RedPajamaWikipedia