text:
- Research article
- Open Access
Limited role for ASC and NLRP3 during in vivo Salmonella Typhimurium infection
BMC Immunology volume 15, Article number: 30 (2014)
The inflammasome is an intracellular protein complex triggered by exposure to intracellular pathogens, its components or other endogenous proteins. It leads to the activation of and subsequent release of proinflammatory cytokines such as IL-1β and IL-18. S. Typhimurium is a Gram-negative intracellular bacterium, which is known to trigger inflammasome assembly via recognition by the cytosolic receptors, NLRP3 and NLRC4 (which act via the adaptor protein, ASC) to induce cell death and cytokine release. We sought to characterize the role of ASC and NLRP3 in two different murine models (typhoid and colitis) of systemic Salmonella infection.
Release of the inflammasome cytokine IL-18 was hampered in Asc−/− but not Nlrp3−/− mice (background C57BL/6) during S. Typhimurium infection. Unexpectedly, neither ASC nor NLRP3 played a significant role in host defense against S. Typhimurium infection, as reflected by equal bacterial counts in WT, Asc−/− and Nlrp3−/− mice at all time points, in both the typhoid and colitis models. Proinflammatory cytokine levels (TNF-α, IL-6) and the extent of hepatic and splenic pathology did not differ between groups in the typhoid model. In the colitis model small differences were seen with regard to splenic and hepatic inflammation, although this was IL-18 independent.
IL-18 release was reduced in Asc−/− but not Nlrp3−/− mice during S. Typhimurium infection. Despite this reduction, bacterial counts, cytokine levels and histological inflammation did not differ between wild-type and knockout mice in either model. Our results reveal a limited role for ASC and NLRP3 during in vivo S. Typhimurium infection despite its role in cytokine maturation.
Invasive Salmonellosis is a global burden with more than 100 million cases per year resulting in over 350,000 deaths –. Salmonella enterica infections, which are the Gram-negative intracellular bacteria responsible for this burden, can result in diverse clinical manifestations. Food-borne non-typhoidal Salmonella (NTS), caused by serovar Typhimurium or Enteriditis, has recently emerged as a prominent cause of bloodstream infection, primarily in African adults and children, with an associated case fatality of up to 25%. HIV, malaria, and malnutrition being important risk factors . Typhoid or enteric fever, caused by the exclusively human serovar Typhi or Paratyphi A, is a bacteremic disease that can result into intestinal perforation, peritonitis, encephalopathy myocarditis and hemodynamic shock –. Antimicrobial resistance for all Salmonella enterica infections is widespread . The variations in the clinical features of infection with these intracellular pathogens relate to differences in the interaction between different Salmonella serovars and the host . A better understanding of host-pathogen interactions in invasive Salmonellosis could explain these diverse clinical manifestations and potentially lead to new therapeutic strategies in order to decrease the considerable morbidity and mortality.
Salmonella spp. are recognized by pattern recognition receptors (PRR), via conserved motifs termed ‘pathogen-associated-molecular-patterns’, resulting in activation of signaling pathways that initiate the inflammatory response ,–. The two most important families of PRRs are the membrane-bound Toll-like receptors (TLRs) and the cytosolic Nod-like receptors (NLRs). Pro-inflammatory cytokines such as interleukin (IL)-6, IL-1β (also called IL-1 F2), IL-18 and tumor necrosis factor (TNF)-α are released during early Salmonella infection. By contrast, interferon (IFN)-γ secretion (triggered by IL-18) plays a central role in the control of persistent infection by affecting the extent of macrophage activation –. Salmonella spp. expresses multiple pathogen-associated-molecular-patterns, most notably type 3 secretion systems (T3SS), flagella, fimbriae, lipopolysaccharide (LPS) and bacterial DNA ,. TLR activation induces the synthesis of pro-IL-1β and pro-IL-18, upon which a second signal is provided by the activation of the intracellular inflammasome and caspase-1 leading to IL-1β and IL-18 processing ,.
The role of the inflammasome in the recognition of Salmonella spp. has been studied extensively using in vitro and in vivo models . Best studied NLRs in the recognition of Salmonella are the pyrin domain containing-3 (NRLP3) and the CARD domain-containing protein-4 (NLRC4), which form inflammasome complexes consisting of caspase-1 and the adaptor protein apoptosis-associated speck-like protein containing a CARD (called PYCARD or ASC) . Both the inflammasome receptors activate caspase-1 in response to S. Typhimurium infection together with endogenous signals, and recruit ASC and caspase-1 into a single cytoplasmatic focus, which subsequently serves as the site for pro-IL-1β processing . Knockout mice lacking functional casp1, casp1-casp11 double knockout mice, or mice deficient in the end product of inflammasome activation (viz., IL-1β and IL-18), do have higher bacterial loads and succumb earlier upon infection with S. Typhimurium ,. It was therefore hypothesized that Nlrp3−/− (and Asc−/− mice especially), having lower levels of inflammasome-processed cytokines, would succumb earlier to infection with S. Typhimurium. On contrary, mice deficient in NLRC4, NLRP3 or ASC were still able to clear S. Typhimurium as efficiently as control mice ,. An explanation given for this discrepancy in literature was that intracellular Salmonella can also induce a Salmonella pathogenicity island (SPI)-1 independent form of lytic cell death via caspase-11 . However, casp11 deficient mice have a phenotype comparable to wild-type (WT) mice, showing a role for caspase-11 in the absence of caspase-1-mediated immunity . Furthermore, pre-growth conditions could have also led to this unexpected outcome, where conditions that favor S PI-1 expression (log-phase bacteria) show minimal roles for the inflammasome, while S PI-2 growth conditions (stationary phase bacteria) show a more significant role for caspase-1 during Salmonella infection . Additionally, NLRC4-dependent bacterial clearance is independent of NLRP3, ASC, IL-18, and IL-1R, which could perhaps be an explanation for the unexpected phenotype of ASC-deficient animals . By contrast, cytokine processing that correlates with the formation of this ASC/caspase-1 focus, ASC-independent inflammasomes containing un-processed but active caspase-1 is also able to initiate rapid cell death . Yet, the relative importance of inflammasome-mediated cytokine maturation and pyroptosis during S. Typhimurium infection still remains to be investigated.
In this study, we examined the in vivo relevance of ASC and NLRP3 during Salmonella infection using two clinically relevant experimental models. All mice received S. Typhimurium orally and either developed a typhoid-like disease (typhoid model) or, when pretreated with streptomycin, developed a non-typhoid salmonellosis (colitis model). We focused on bacterial growth, systemic cytokine release and organ pathology caused by S. Typhimurium using log-phase bacteria.
Experiments were carried out in accordance with the Dutch Experiment on Animals Act and approved by the Animal Care and Use Committee of the University of Amsterdam (Permit number: DIX102113, DIX102114).
Eight- to 12-week-old male Asc−/− and Nlrp3−/− mice, backcrossed 9 times to a C57BL/6 genetic background, were generated as described  and bred in the animal facility of the Academic Medical Center (Amsterdam, the Netherlands). Pathogen-free C57BL/6 WT mice were purchased from Charles River Laboratories Inc. (Maastricht, the Netherlands). Age- and sex-matched animals were used in all experiments and were housed in rooms with a controlled temperature a 12-h light–dark cycle, and were acclimatized for 2 weeks before the experiments. All experimental procedures and animal handling were done during the light cycle.
Experimental infection and design
For preparation of the inoculum Salmonella Typhimurium strain 14028 was used (ATCC, LGC Standards GmbH, Wesel, Germany). Stock bacteria were streaked from frozen aliquots into 50 ml Luria broth for overnight incubation at 37°C in a 5% CO2 incubator. Thereafter, a 1 ml portion for 1:50 dilution was transferred to fresh Luria broth and grown for approximately 2 h to mid-logarithmic phase (OD 0.4). Bacteria were harvested by centrifugation at 1500 × g for 10 min, washed and resuspended in sterile isotonic Hank’s Buffered Salt Solution (HBSS) at a concentration of 107 cfu/ml S. Typhimurium. Inoculum size was verified retrospectively by serial 10-fold dilutions on blood agar (BA). Two different Salmonella infection models were used: mice were starved for 12 h (typhoid model) or pre-treated with streptomycin (Rotexmedica GmbH, Trittau, Germany) 7.5 mg/100 μl HBSS per os (colitis model), and inoculated orally with 106 cfu/ml S. Typhimurium in 100 μL HBSS as described previously –. At 2 and 5 days after infection (typhoid model) and 4 days (colitis model), mice were anesthetized with Hypnorm (Janssen Pharmaceutica, Beerse, Belgium) and midazolam (Roche, Basel, Switzerland), and then sacrificed by bleeding from the inferior vena cava. Blood was drawn into heparinized tubes and organs (Mesenterial lymph nodes (MLN), liver, spleen) were harvested and homogenized at 4°C in four volumes of sterile saline using a tissue homogenizer (Biospec Products, Bartlesville, UK). Death was confirmed by cutting the diaphragm. Bacterial loads were determined by serial ten-fold dilutions on BA incubated at 37°C for 16 h. Homogenates were centrifuged at 1500 × g at 4°C for 10 min, and supernatants were stored at −20°C pending assay.
IL-18 (Medical and Biological Laboratories, Nagoya, Japan), IL-1β and TNF-α (R&D, Minneapolis, MN) concentrations were measured by enzyme-linked immunosorbent assays. IFN-γ, MCP-1, IL-6, IL-10 and IL-12 were measured by cytometric bead array (BD Biosciences, San Jose, CA) in accordance with the manufacturer’s recommendations. AST, ALT and LDH were measured in plasma with spectrophotometry (Roche Diagnostics).
Liver, spleens, MLN, terminal ileum and cecum were harvested after sacrifice, fixed in 10% formalin and embedded in paraffin for histology. Sections of 4 μm were stained with haematoxylin–eosin, and read by a pathologist who was blinded to the groups. To score liver inflammation and damage, the entire slide surface was analyzed with respect to the following parameters: area of liver with parenchymal inflammation, necrosis and/or abscess formation, portal inflammation and thrombus formation. Each parameter was graded on a scale of 0 to 4 (0: absent; 1: mild; 2: moderate; 3: severe; 4: very severe). Thrombi were scored as follows: 0: no thrombi; 1: 1–4 thrombi; 2: 5–9 thrombi; 3: 10–15 thrombi; 4: more than 15. The total liver inflammation score was expressed as the sum of the scores for each parameter, the maximum being 16. Spleen sections were scored for inflammation, necrosis/abscess formation, and thrombus formation using the scales given above. The maximum total spleen inflammation score was 12. In both mouse models the cecum, colon and terminal ileum were assessed for neutrophil infiltration, edema and disruption of the epithelium to determine whether there was a colitis or gut infection present as is expected in the colitis model (streptomycin pre-treatment group).
Values are expressed as mean with standard error of the mean unless stated otherwise. Differences between groups were analyzed by Mann–Whitney U test. These analyses were performed using GraphPad Prism version 5.0, GraphPad Software (San Diego, CA). Values of p < 0.05 were considered statistically significant.
Murine typhoid model for invasive salmonellosis
In order to study the in vivo role of ASC and NLRP3 in invasive salmonellosis, we used a murine typhoid model for severe Salmonella infection . Mice were infected with S. Typhimurium strain S14208 per os. This model mimics the pathology of untreated patients presenting with severe typhoid fever and is characterized by advanced liver and spleen inflammation and capillary thrombus formation in the Peyer’s patches, which may lead to hemorrhage, necrosis, ulceration and intestinal perforation . The onset of clinical symptoms and the end stage of disease were dependent on the dose administered. Our pilot experiments showed that an oral dosage of 106 CFU S. Typhimurium resulted in a mortality rate of over 90% (LD90) within 2 weeks. When the dosage was increased to 108 CFU S. Typhimurium most mice (LD75) died within a week. When mice became clinically ill (as indicated by ruffled fur, behavioral changes and decreased activity) bacterial counts in blood had increased dramatically from 101 to 105 cfu/ml at 2 and 5 days post-infection respectively, with a marked rise in proinflammatory cytokines, most notably TNF-α, IFN-γ and IL-6, in the systemic compartment.
IL-18 is elevated during S. Typhimurium infection in WT mice but completely abolished in Asc−/− mice
Inoculation of WT mice with S. Typhimurium resulted in a rapid increase in IL-18 plasma concentrations in the typhoid model (from 162 pg/ml at day 2, to 627 pg/ml 5 days post infection; p = 0.02), corresponding with inflammasome activation (Figure 1A). In Asc−/− mice IL-18 remained undetectable during the course of infection (Figure 1A). Nlrp3−/− mice, however, expressed equal plasma levels of IL-18 after infection at all time points when compared to WT mice (Figure 1E), indicating that IL-18 production is independent of NLRP3 during experimental S. Typhimurium infection in vivo. IL-1β levels remained undetectable in plasma from WT, Asc−/− and Nlpr3−/− mice after infection with S. Typhimurium (data not shown).
Asc−/− and Nlrp3−/− mice display equal bacterial counts in all organs upon infection with S. Typhimurium
Having established that IL-18 is upregulated during murine S. Typhimurium infection, we infected WT, Asc−/− and Nlrp3−/− mice with S. Typhimurium (inoculum 106 cfu, typhoid model) and sacrificed them after 2 and 5 days (i.e., before the first deaths occurred) to determine the bacterial loads in MLN (the primary site of the infection once invading bacteria have penetrated the mucosal barrier), liver, spleen and blood in order to evaluate bacterial loads and dissemination to distant body sites. Relative to WT mice, both Asc−/− and Nlrp−/− mice displayed equal bacterial loads in MLNs, liver, spleen and blood at 2 and 5 days after infection (Figure 1B-D, F-H; spleen not shown).
Systemic inflammatory non-inflammasome related cytokine release during in vivo S. Typhimurium infection is ASC- and NLRP3-independent
We next examined the impact of ASC and NLRP3 deficiency on the host cytokine response to S. Typhimurium in the typhoid mouse model. We measured concentrations of TNF-α, IFN-γ, MCP-1, IL-6, IL-10 and IL-12p70 in plasma of Asc−/−, Nlrp3−/− and WT mice 2 and 5 days after infection. Two days post-infection most systemic cytokine levels were still below the detection limit (data not shown). After 5 days of infection, however, plasma levels TNF-α, monocyte chemo attractive protein (MCP)-1, IL-6, IL-10 and IL-12 were markedly elevated in all WT mice (Figure 2). Remarkably, neither ASC nor NLRP3 deficiency influenced systemic inflammation: no differences were found in any of the cytokine levels measured when comparing WT with Asc−/− or Nlrp3−/− mice, except for a slight increase in IL-12 in Asc−/− mice 5 days post-infection which could be a compensation for the lack of IL-18 observed in these mice. Surprisingly, plasma IFN-γ levels (Figure 2B) did not differ between WT and Asc−/− mice during S. Typhimurium infection (2 and 5 days post-infection) despite the observed marked differences in release of IL-18, which is a key trigger for IFN-γ release via NK and T-cells, dendritic and other phagocytic cells during Salmonella infection ,.
Severe hepatic inflammation and distant organ injury during S. Typhimurium infection are ASC- and NLRP3-independent
Next, we semi-quantitatively scored for the extent of inflammation histological samples of liver and spleen obtained 2 and 5 days after infection to further evaluate the role of ASC and NLRP3 in systemic in vivo host defense against S. Typhimurium in the typhoid model. During S. Typhimurium infection, hepatic inflammation was characterized by significant inflammation with necrosis, abscess formation, portal inflammation and thrombus formation, which was most prominent 5 days after infection (Figure 3). No differences were observed between WT, Asc−/− and Nlrp3−/− mice (Figure 3) in pathology scores, which corresponded to the results of bacterial burdens and cytokine profiles. Spleen inflammation scores were similar in all three mice strains studied at all time points (data not shown). Consistent with these pathology data, systemic markers of organ injury were significantly elevated during S. Typhimurium infection, reflecting increased hepatocellular injury (indicated by elevated levels of Aspartate aminotransferase (AST) and alanine transaminase (ALT)) and tissue damage (indicated by elevated lactate dehydrogenase (LDH) levels). However, these effects were not influenced by ASC or NLRP3 deficiency (Figure 4).
Limited role for both ASC and NLRP3 in systemic host defense against S. Typhimurium in the colitis model
From the previous studies, which were performed in the typhoid model of Salmonella infection, we concluded that ASC and NLRP3 only play a very limited role in the systemic immune response to S. Typhimurium infection. To investigate whether these findings were dependent on the model chosen, we examined the role of these inflammasome components in the colitis model of Salmonella infection, in which mice were pre-treated orally with streptomycin. Similar to the typhoid model, Asc−/− mice displayed undetectable IL-18 levels, whereas plasma IL-18 concentrations in Nlrp3−/− mice did not differ from those in WT mice (Figure 5A). In contrast to the typhoid model, wherein mice do not develop marked inflammation of the cecum and colon, the colitis model results in a profound local inflammatory response with severe colitis and abscess formation after which Salmonella spreads systemically (Additional file 1). We found no difference when comparing WT, Asc−/− and Nlrp3−/− mice in our colitis model with respect to bacterial counts and dissemination towards the liver and the systemic compartment 4 days after infection (Figure 5B-D), which corresponds to our findings in the typhoid model; the only exception being a slight increase in bacterial counts in the MLNs of Asc−/− mice when compared to MLNs from WT mice. No differences were observed in plasma TNF-α, MCP-1, IL-6, IL-10 and IL-12 levels between WT, Asc−/− and Nlrp3−/− mice after infection with S. Typhimurium (Figure 6). Of note, in the colitis model, IFN-γ correlated to IL-18 production and was reduced in the Asc−/− mice when compared to WT mice (234 ± 66 to 1831 ± 426 pg/ml, p = 0.001; Figure 6B). A difference between liver and spleen pathology is seen when comparing the typhoid and colitis models: while the typhoid fever model is characterized by marked hepatic necrosis, this was not regularly observed in the colitis model, while marked hepatic inflammatory cell influx is seen in both models (Figure 5J-L) Moreover, in the colitis model Nlrp3−/− (and to a lesser extent, Asc−/− mice) displayed slightly increased spleen and liver parenchyma inflammation with corresponding elevated AST, albeit not ALT, levels when compared to WT mice. This may suggest that the differences in inflammation or damage caused by S. Typhimurium may be IL-18 independent.
In the present study we aimed to characterize the in vivo relevance of the central inflammasome molecules ASC and NLRP3 in two different murine models of systemic Salmonella infection. Although in vitro studies confirmed that the inflammasome is activated during S. Typhimurium infection, we here show that ASC and NLRP3 individually are dispensable for the development of innate immunity against the pathogen. In the murine typhoid model, in which mice were not pretreated with streptomycin prior to oral inoculation with S. Typhimurium, equal bacterial counts were seen in WT, Asc−/− and Nlrp3−/− mice after infection in all organs (MLNs, liver, spleen, blood) at all time points. In line with the bacterial loads, proinflammatory cytokine levels, markers for tissue damage and organ pathology did not differ between groups. These results were replicated in a colitis model of S. Typhimurium infection, in which mice were pretreated with streptomycin prior to oral inoculation, were we saw an equally limited role for both ASC and NLRP3 in systemic host defense against S. Typhimurium. Taken together, the present results reveal a surprisingly limited role for ASC and NLRP3 during in vivo S. Typhimurium infection when dissecting different local and systemic compartments, especially given the fact that Salmonella infection is generally believed to be intracellular and that the inflammasome has previously been described to be crucial for the host defense against other intracellular pathogens .
NLRP3 can be triggered via terminal signals such as lysosomal rupture and the release of cathepsins and potassium, and the production of reactive oxygen species. The precise bacterial trigger for NLRP3 in S. Typhimurium infection remains unclear, since it has been demonstrated that Salmonella pathogenicity island-2 T3SS mutants (which lack the capacity to replicate intracellularly) are still detected by NLRP3 ,,. More recently, it was shown that NLRP3 inflammasome activation is bacterial RNA-mediated , which might explain the NLRP3-dependent sensing of these mutants. Viable S. Typhimurium can trigger both the NRLP3 and NLRC4 inflammasomes in vitro, resulting in activation of caspase-1, without involvement of other inflammasomes. The release of IL-1β was shown to be completely dependent on the combined function of the NLRP3 and NLRC4 inflammasomes and blocking either one lead to partially decreased IL-1β release . The adaptor molecule ASC is a crucial component of both the NLRP3 and NLRC4 inflammasomes as it can bridge the pyrin and CARD domain of these NRLs to caspase-1, which is necessary for its activation . Asc−/− bone-marrow derived macrophages stimulated with S. Typhimurium in vitro had defects in both the NLRP3 and NLRC4 inflammasomes with regard to releasing mature IL-1β . Although these studies all underscore the potential role for the inflammasome during Salmonella infection in vitro, previous in vivo studies were not as evident.
During S. Typhimurium infection IL-1β is reported to be critical for the intestinal phase of the disease, while IL-18 is important for resistance to systemic infection but not the early gastrointestinal phase of infection . Mice lacking both Nlrp3 and Nlrc4 genes showed an increased susceptibility to S. Typhimurium infection, similar to caspase-1−/− or Il-18−/− mice, whereas mice lacking either NLRP3 or NLRC4 did not succumb earlier to infection, which was ascribed to the ability of NLRP3 and NLRC4 to be engaged by distinct signals ,,. In previous studies using a typhoid infection model, Asc−/− mice were shown to have comparable bacterial loads (MLNs, spleen and liver) relative to WT mice when challenged with a different Salmonella strain (S. Typhimurium SL1344), while their serum IL-18 levels remained low, albeit not significantly different ,. It was therefore speculated that ASC may play a role in vivo in the maturation of cytokines, whereas it is not essential for restricting bacterial growth, suggesting that additional ASC-independent pathways could be involved in activating caspase-1 in response to NLRC4 and NLRP3 activation . Indeed, it was shown that NLRC4 could also activate caspase-1 even in the absence of ASC, to induce pyroptosis, ASC however remains to be crucial for cytokine maturation during Salmonella infection ,. Furthermore, intracellular Salmonella could induce a form of lytic cell death via caspase-11 , which was IFN-γ-mediated ,. To study the in vivo role of the ASC and NLRP3 molecules in more depth we here made use of two different lethal S. Typhimurium models modeling typhoid and colitis disease. We showed that IL-18 release in plasma of Asc−/− mice was significantly decreased in both models and previous studies have suggested that impaired IL-18 release could enhance S. Typhimurium susceptibility in mice . Surprisingly, we were unable to find a role for either ASC or NLRP3 in the host defense against S. Typhimurium infection in the typhoid model: no differences were found in bacterial counts in any organ (MLNs, liver, spleen and blood) at all time points. Furthermore, proinflammatory cytokine levels (TNF-α, IL-6, IL-10, MCP-1, IFN-γ), markers for hepatocellular injury (AST, ALT), cell damage in general (LDH) and organ pathology (liver, spleen) did not differ between groups. In a similar study using a Burkholderia pseudomallei infection model, it was noted that IL-18 release was drastically reduced in Asc−/− and Nlrp3−/− mice, although it was still detectable in these mice at higher levels than uninfected mice, leading to the conclusion that inflammasome-independent production of IL-18 may be sufficient to provide some level of protection against infection with low dosages of Burkholderia pseudomallei. This could be an explanation for the phenotype observed in the Nlrp3−/− mice, which was comparable to WT mice, but not for the Asc−/− mice, as in both our infection models the IL-18 levels of Asc−/− mice remained below detection limit independent of the stage of infection.
Although no differences were observed between bacterial loads in WT and Asc−/− or Nlrp3−/− mice in the typhoid model, small differences in the streptomycin pre-treated group were seen with regard to bacterial loads (MLN) and splenic and hepatic inflammation between groups. A potential explanation for the different roles played by ASC in both models with regards to hepatocellular injury could be the marked difference in IFN-γ release: where IFN-γ was not reduced in the typhoid model, it was reduced in Asc−/− mice in the colitis model. In Salmonella- infected mice pre-treated with streptomycin, IFN-γ seems to coordinate T-cell responses and the anti-microbial activity of phagocytes . Il-18−/− mice show an increased susceptibility to S. Typhimurium infection . Interestingly, in vivo IL-18 neutralization causes a marked decrease in circulating IFN-γ levels during Salmonella infection . It could therefore be hypothesized that the increased susceptibility to S. Typhimurium of Il-18−/− mice could in part be caused by the low circulating IFN-γ levels and not by absence of IL-18 per se. The normal IFN-γ response in the typhoid model could potentially trigger the described ASC-independent pathway via caspase-11 and IFN-γ . It has to be noted, however, that this does not explain the observed differences between IFN-γ levels in the typhoid- and colitis model in Asc−/− mice and the increased hepatocellular injury inflicted upon Nlrp3−/− mice despite lower but not significantly different IFN-γ levels in the colitis model.
Our study has several limitations. Pre-growth conditions of Salmonella could have influenced our results as it determines expression of S PI-1 and S PI-2 expression among other virulence factors necessary for inflammasome activation ,. We choose to use log-phase bacteria in our models according to standard methods in order to induce experimental infection in mice. Furthermore, the NLRC4 receptor has been described to play an important role during Salmonella infection ,; adding a functional knockout mice to our experimental protocol could have given additional insights into the role of this inflammasome receptor in the typhoid and colitis model. It should be noted however that at present 22 human NLRs have been described and 34 mouse NLRs . A plausible alternative interpretation of our work might be the presence of notable redundancy in the mouse system of NLRs so that one or more NLRs may be able to take over the role of NLRP3 in knockout mice (but not necessarily in humans), and that a double (or triple) knockout may be necessary in order to see a phenotype (Asc/Il-18 double KO for instance). Indeed, Broz et al. recently showed that Nlrp3/Nlrc4 double knockout mice have a markedly increased susceptibility for invasive salmonellosis when compared to WT controls .
In this work, we tried to further detail the role of key-inflammasome proteins ASC and NLRP3 during in vivo Salmonella infection. Inflammasome pathways are intensely complex and have proven to show immense redundancy. Indeed, our results reveal a limited role for both ASC and NLRP3 during in vivo S. Typhimurium infection, which is remarkable given the central role attributed to the inflammasome in the host defense against intracellular bacteria. In the future, it would be of great interest to study these proteins in humans with invasive salmonellosis or to make use of KO mice with complete blockage of all inflammasomes important in Salmonella pathogenesis.
Pattern recognition receptor
- S. enterica :
Pattern recognition receptor
Tumor necrosis factor-alpha
Type 3 secretion system
NOD-like receptor pyrin domain containing 3
NOD-like receptor, CARD domain-containing protein 4
Apoptosis-associated speck-like protein containing a CARD
- SPI Salmonella:
Hank’s buffered salt solution
Mesenteric lymph nodes
Monocyte chemo attractive protein-1
Crump JA, Mintz ED: Global trends in typhoid and paratyphoid Fever. Clin Infect Dis. 2010, 50 (2): 241-246. 10.1086/649541.
Feasey NA, Dougan G, Kingsley RA, Heyderman RS, Gordon MA: Invasive non-typhoidal salmonella disease: an emerging and neglected tropical disease in Africa. Lancet. 2012, 379 (9835): 2489-2499. 10.1016/S0140-6736(11)61752-2.
de Jong HK, Parry CM, van der Poll T, Wiersinga WJ: Host-pathogen interaction in invasive salmonellosis. PLoS Pathog. 2012, 8 (10): e1002933-10.1371/journal.ppat.1002933.
Dougan G, John V, Palmer S, Mastroeni P: Immunity to salmonellosis. Immunol Rev. 2011, 240 (1): 196-210. 10.1111/j.1600-065X.2010.00999.x.
Parry CM, Hien TT, Dougan G, White NJ, Farrar JJ: Typhoid fever. N Engl J Med. 2002, 347 (22): 1770-1782. 10.1056/NEJMra020201.
Parry CM, Threlfall EJ: Antimicrobial resistance in typhoidal and nontyphoidal salmonellae. Curr Opin Infect Dis. 2008, 21 (5): 531-538. 10.1097/QCO.0b013e32830f453a.
Kawai T, Akira S: The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol. 2010, 11 (5): 373-384. 10.1038/ni.1863.
Schroder K, Tschopp J: The inflammasomes. Cell. 2010, 140 (6): 821-832. 10.1016/j.cell.2010.01.040.
Broz P, Monack DM: Molecular mechanisms of inflammasome activation during microbial infections. Immunol Rev. 2011, 243 (1): 174-190. 10.1111/j.1600-065X.2011.01041.x.
Butler T, Ho M, Acharya G, Tiwari M, Gallati H: Interleukin-6, gamma interferon, and tumor necrosis factor receptors in typhoid fever related to outcome of antimicrobial therapy. Antimicrob Agents Chemother. 1993, 37 (11): 2418-2421. 10.1128/AAC.37.11.2418.
Keuter M, Dharmana E, Gasem MH, van der Ven-Jongekrijg J, Djokomoeljanto R, Dolmans WM, Demacker P, Sauerwein R, Gallati H, van der Meer JW: Patterns of proinflammatory cytokines and inhibitors during typhoid fever. J Infect Dis. 1994, 169 (6): 1306-1311. 10.1093/infdis/169.6.1306.
Raffatellu M, Chessa D, Wilson RP, Tukel C, Akcelik M, Baumler AJ: Capsule-mediated immune evasion: a new hypothesis explaining aspects of typhoid fever pathogenesis. Infect Immun. 2006, 74 (1): 19-27. 10.1128/IAI.74.1.19-27.2006.
Thompson LJ, Dunstan SJ, Dolecek C, Perkins T, House D, Dougan G, Nguyen TH, Tran TP, Doan CD, Le TP, Nguyen TD, Tran TH, Farrar JJ, Monack D, Lynn DJ, Popper SJ, Falkow S: Transcriptional response in the peripheral blood of patients infected with Salmonella enterica serovar Typhi. Proc Natl Acad Sci U S A. 2009, 106 (52): 22433-22438. 10.1073/pnas.0912386106.
Monack DM, Bouley DM, Falkow S:Salmonella typhimurium persists within macrophages in the mesenteric lymph nodes of chronically infected Nramp1+/+mice and can be reactivated by IFNgamma neutralization. J Exp Med. 2004, 199 (2): 231-241. 10.1084/jem.20031319.
Kupz A, Guarda G, Gebhardt T, Sander LE, Short KR, Diavatopoulos DA, Wijburg OL, Cao H, Waithman JC, Chen W, Fernandez-Ruiz D, Whitney PG, Heath WR, Curtiss R, Tschopp J, Strugnell RA, Bedoui S: NLRC4 inflammasomes in dendritic cells regulate noncognate effector function by memory CD8(+) T cells. Nat Immunol. 2012, 13 (2): 162-169. 10.1038/ni.2195.
Mastroeni P, Clare S, Khan S, Harrison JA, Hormaeche CE, Okamura H, Kurimoto M, Dougan G: Interleukin 18 contributes to host resistance and gamma interferon production in mice infected with virulent Salmonella typhimurium. Infect Immun. 1999, 67 (2): 478-483.
Miao EA, Leaf IA, Treuting PM, Mao DP, Dors M, Sarkar A, Warren SE, Wewers MD, Aderem A: Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria. Nat Immunol. 2010, 11 (12): 1136-1142. 10.1038/ni.1960.
Broz P, Newton K, Lamkanfi M, Mariathasan S, Dixit VM, Monack DM: Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella. J Exp Med. 2010, 207 (8): 1745-1755. 10.1084/jem.20100257.
Raupach B, Peuschel SK, Monack DM, Zychlinsky A: Caspase-1-mediated activation of interleukin-1beta (IL-1beta) and IL-18 contributes to innate immune defenses against Salmonella enterica serovar Typhimurium infection. Infect Immun. 2006, 74 (8): 4922-4926. 10.1128/IAI.00417-06.
Broz P, Ruby T, Belhocine K, Bouley DM, Kayagaki N, Dixit VM, Monack DM: Caspase-11 increases susceptibility to Salmonella infection in the absence of caspase-1. Nature. 2012, 490: 288-291. 10.1038/nature11419.
Lara-Tejero M, Sutterwala FS, Ogura Y, Grant EP, Bertin J, Coyle AJ, Flavell RA, Galan JE: Role of the caspase-1 inflammasome in Salmonella typhimurium pathogenesis. J Exp Med. 2006, 203 (6): 1407-1412. 10.1084/jem.20060206.
Broz P, von Moltke J, Jones JW, Vance RE, Monack DM: Differential requirement for Caspase-1 autoproteolysis in pathogen-induced cell death and cytokine processing. Cell Host Microbe. 2010, 8 (6): 471-483. 10.1016/j.chom.2010.11.007.
Sutterwala FS, Ogura Y, Szczepanik M, Lara-Tejero M, Lichtenberger GS, Grant EP, Bertin J, Coyle AJ, Galan JE, Askenase PW, Flavell RA: Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity. 2006, 24 (3): 317-327. 10.1016/j.immuni.2006.02.004.
Carter PB, Collins FM: The route of enteric infection in normal mice. J Exp Med. 1974, 139 (5): 1189-1203. 10.1084/jem.139.5.1189.
Barthel M, Hapfelmeier S, Quintanilla-Martinez L, Kremer M, Rohde M, Hogardt M, Pfeffer K, Russmann H, Hardt WD: Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host. Infect Immun. 2003, 71 (5): 2839-2858. 10.1128/IAI.71.5.2839-2858.2003.
Hapfelmeier S, Hardt WD: A mouse model for S. typhimurium-induced enterocolitis. Trends Microbiol. 2005, 13 (10): 497-503. 10.1016/j.tim.2005.08.008.
Santos RL, Zhang S, Tsolis RM, Kingsley RA, Adams LG, Baumler AJ: Animal models of Salmonella infections: enteritis versus typhoid fever. Microbes Infect. 2001, 3 (14–15): 1335-1344. 10.1016/S1286-4579(01)01495-2.
Kufer TA, Sansonetti PJ: NLR functions beyond pathogen recognition. Nat Immunol. 2011, 12 (2): 121-128. 10.1038/ni.1985.
Sander LE, Davis MJ, Boekschoten MV, Amsen D, Dascher CC, Ryffel B, Swanson JA, Muller M, Blander JM: Detection of prokaryotic mRNA signifies microbial viability and promotes immunity. Nature. 2011, 474 (7351): 385-389. 10.1038/nature10072.
Martinon F, Mayor A, Tschopp J: The inflammasomes: guardians of the body. Annu Rev Immunol. 2009, 27: 229-265. 10.1146/annurev.immunol.021908.132715.
Mayer-Barber KD, Barber DL, Shenderov K, White SD, Wilson MS, Cheever A, Kugler D, Hieny S, Caspar P, Nunez G, Schlueter D, Flavell RA, Sutterwala FS, Sher A: Caspase-1 independent IL-1beta production is critical for host resistance to mycobacterium tuberculosis and does not require TLR signaling in vivo. J Immunol. 2010, 184 (7): 3326-3330. 10.4049/jimmunol.0904189.
Ceballos-Olvera I, Sahoo M, Miller MA, Del Barrio L, Re F: Inflammasome-dependent pyroptosis and IL-18 protect against Burkholderia pseudomallei lung infection while IL-1beta is deleterious. PLoS Pathog. 2011, 7 (12): e1002452-10.1371/journal.ppat.1002452.
Kaiser P, Diard M, Stecher B, Hardt WD: The streptomycin mouse model for Salmonella diarrhea: functional analysis of the microbiota, the pathogen’s virulence factors, and the host’s mucosal immune response. Immunol Rev. 2012, 245 (1): 56-83. 10.1111/j.1600-065X.2011.01070.x.
Mariathasan S, Newton K, Monack DM, Vucic D, French DM, Lee WP, Roose-Girma M, Erickson S, Dixit VM: Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature. 2004, 430 (6996): 213-218. 10.1038/nature02664.
Ting JP, Lovering RC, Alnemri ES, Bertin J, Boss JM, Davis BK, Flavell RA, Girardin SE, Godzik A, Harton JA, Hoffman HM, Hugot JP, Inohara N, Mackenzie A, Maltais LJ, Nunez G, Ogura Y, Otten LA, Philpott D, Reed JC, Reith W, Schreiber S, Steimle V, Ward PA: The NLR gene family: a standard nomenclature. Immunity. 2008, 28 (3): 285-287. 10.1016/j.immuni.2008.02.005.
HdJ received unrestricted funding for this project from the Academic Medical Center (AMC PhD Scholarship). JW: The Netherlands Organisation for Scientific Research (NWO; VENI grant number 91610008) and The Netherlands Organisation for Health Research and Development (ZonMw; Clinical Fellowship grant number 90700424). We thank Richard A. Flavell, Yale University School of Medicine, for generously providing us the Asc−/− and Nlrp3−/− mouse strains. We also would like to thanks Marieke van den Brink and Joost Daalhuisen for technical assistance with mouse handling.
The authors declare that they have no competing interests.
Conceived and designed the experiments: HdJ, JvD, TvdP, and JW. Performed the experiments: HdJ, GK and MvL. Analysed the data: HdJ, MvL, GK, JR, JW. Wrote the paper: HdJ, TvdP, JW. All authors approved the final version for submission.
Electronic supplementary material
Additional file 1:Severe intestinal pathology seen in mice pretreated with streptomycin post infection with S. Typhimurium. Wild-type (WT) mice were starved for 12 h (typhoid fever model) or pretreated with streptomycin (colitis model) before infection with S. Typhimurium (106) per os and sacrificed post-infection to assess intestinal pathology. Representative photographs of haemotoxylin and eosin (HE) stained slides are displayed of the cecum of the typhoid model (A-C) and colitis model (D-F) 4–5 days post infection. Original magnifications. ×4 (A, D), ×10 (B, E), and × 20 (C, F). Severe intestinal pathology characterized by neutrophil infiltration, edema and extensive destructive ulceration of the mucosa is seen in mice pretreated with streptomycin (colitis model) but not in a typhoid fever model. Arrowheads indicate severe colitis in mice pretreated with streptomycin accompanied by crypt abscesses (*). (PDF 6 MB)
Authors’ original submitted files for images
About this article
Cite this article
De Jong, H.K., Koh, G.C., van Lieshout, M.H. et al. Limited role for ASC and NLRP3 during in vivo Salmonella Typhimurium infection. BMC Immunol 15, 30 (2014). https://doi.org/10.1186/s12865-014-0030-7
- Salmonella Typhimurium
- Host-pathogen interactions

domain: biology


text:
Love Is........ - Poem by Tressa Carliss
Love is trust between two people
A compromise, a promise, an agreement
To be honest at all times no matter what
So what is love when based on a lie.
When based on a lie love is....
A pathetic attempt to date someone
A tragedy just waiting to happen
A lie all it's own.
When based on a lie love is not love
When based on a lie, a marriage in turn is a lie
When based on a lie, love does not exist
When based on lie, you do not exist.
Comments about Love Is........ by Tressa Carliss
Read this poem in other languages
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Still I Rise
The Road Not Taken
If You Forget Me
Edgar Allan Poe
Stopping By Woods On A Snowy Evening
I Do Not Love You Except Because I Love You

domain: artistic


text:
Premier League winner Marc Albrighton joins Albion with a wealth of top-level experience to his name.
The 33-year-old was born in Tamworth and came through at Aston Villa, making his competitive debut during a UEFA Cup match at CSKA Moscow in February 2009.
A maiden Premier League appearance came five months later at the beginning of the 2009/10 campaign, and he went on to play a total of 101 matches across all competitions for Villa – scoring nine goals.
- Did you know? Marc scored the Premier League’s 20,000th goal while at Villa Park, winning a £20,000 cheque from then-league sponsors Barclays and donating it to local charity Acorns Children’s Hospice.
But it was with his next club, Leicester City, where Albrighton’s career reached new heights. Joining at the start of the 2014/15 term, Marc was key in the Foxes’ memorable ‘great escape’ under Nigel Pearson before they went on to complete their famous Premier League triumph.
Marc featured in every single league game on the way to Leicester lifting the title, and was singled out by manager Claudio Ranieri for his work-rate, defensive ability and quality going forward, particularly through crosses and set-pieces.
- Did you know? Albrighton cited England legend David Beckham as one of his idols growing up.
Capable of playing in a number of positions, the former England-U21 international netted Leicester’s first ever Champions League goal during a 3-0 victory over Club Brugge in September 2016.
He was an important part of the Foxes team that reached the last-eight in Europe, which was then backed up by consecutive ninth-place finishes in the Premier League in 2017/18 and 2018/19.
Further European experience, in the form of Europa League football, soon came after a top-five finish the following year.
And he came off the bench at Wembley in May 2021 when Leicester won the FA Cup, as well as the FA Community Shield a few months later, adding further medals to his impressive individual collection.
Albrighton hasn’t been short of minutes this season, either, playing a total of 11 times – including five appearances in the last month across the league and both domestic cup competitions.

domain: news


text:
The repercussions of the Uri terror attacks are getting intense. After Maharashtra Navnirman Sena (MNS) warned all Pakistani artistes in India to leave the country or face consequences, Indian Motion Picture Producers Association has also imposed a ban on Pakistani actors working in India.
Jab We Met (Mauja Hi Mauja) FULL SONG *HQ*
Oct 26 2007 Jab We Met Mauja Hi FULL SONG *HQ* Jab We Met Aaoge Tum FULL SONG *HQ* Aao Milo Chalo Kareena Kapoor Shahid Imtiaz Ali Pritam Three Idiots Main Aur Mrs Khanna Kambakkht Ishq Billu Barber Luck By Chance Milenge Golmaal Returns Roadside Romeo Tashan Kya Love Story Hai Dil Bole Hadippa Paathshala Kaminey Kismat Konnection Aaj Kal Blue
Superstar Shah Rukh Khan is currently in Europe shooting for Imtiaz Ali’s film which is tentatively title ‘The Ring’. The actor is starring opposite his ‘Rab Ne Banadi Jodi’ co-star Anushka Sharma in the movie. The shooting began in Prague after which the team headed to Amsterdam, and now
Bollywood superstar Shah Rukh Khan believes that he can fly and he almost did it. The 50-year-old actor, who displayed his superpowers on-screen in the film ‘Ra.One’, has shared a video of him ‘flying’ on Instagram. In the video, Shah Rukh is seen ‘flying’, or rather sliding down the raili

domain: celebrity


text:
International pop star Ariana Grande is finally visiting her fans in Seoul. On August 15, the artist will perform at Gocheok Sky Dome in Seoul, as a part of her Dangerous Woman world tour.
Grande is in the middle of her 8-month-long world tour, visiting cities all around the world since February 3. Despite the recent Manchester Arena bombing which caused 22 deaths at the concert, the artist is carrying on with her tour schedule, in addition to holding a benefit concert to support those who were attacked.
Grande’s upcoming Seoul concert is hosted as a part of Hyundai Card’s Culture Project, which invites young and leading artists from all over the world. The project has been acclaimed for bringing some of the best internationally renowned artists — and not just limited to the musical artists, but also encompassing those from various other genres of art such as architecture and acting. The Korean credit card company has also recently presented Coldplay’s A Head Full of Dreams concert tour.
“The upcoming concert will be a precious opportunity for fans in Korea to witness Ariana’s ability as she is growing as the world’s greatest diva,” introduces Hyundai Card through a press release.
Tickets for Grande’s Seoul concert will be available on June 26 for Hyundai Card holders and June 27 for the general public. The price ranges from 99,000 KRW to 143,000 KRW.
By Heewon Kim


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Gina1960 said:Cheryl are the leaves velvety? I think its the same as mine. Hafta look it up. I used to have so many different costus! Only have a few now.
Gina1960 said:Then I would say C. curvibrachateaus
Gina1960 said:I just looked up my Costus in the old plant files I have of some of my plants. Wish I had them for ALL of my plants. Mine is the Dwarf Costus Barbatus. I got it from Aloha Tropicals way back in the wayback ,c2006. Used to have a lot of COstus like the chalice ones and the lollipop ones but no longer have them.


RPC server port is always a random one. In fact, the problem is in RpcConfiguration.HIVE_SPARK_RSC_CONFIGS which doesn't include SPARK_RPC_SERVER_PORT.
I've found this issue while trying to make hive-on-spark running inside docker.
HIVE_SPARK_RSC_CONFIGS is called by HiveSparkClientFactory.initiateSparkConf > SparkSessionManagerImpl.setup and the latter call SparkClientFactory.initialize(conf) which initialize the rpc server. This RPCServer is then used to create the sparkClient which use the rpc server port as --remote-port arg. Since initiateSparkConf ignore SPARK_RPC_SERVER_PORT, then it will always be a random port.


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text:
Taylor Lautner and girlfriend Taylor Dome confirm romance in a loved up Halloween photo
It seems it's only official once it's been posted on Instagram.
And that's exactly what Taylor Lautner did to confirm his relationship with Taylor Dome on Monday.
The doting couple was seen sporting matching Space Jam costumes for their Halloween post, which sees Taylor passionately planting a kiss on his girlfriend's cheek.
It's official: Taylor Lautner confirmed his relationship with Taylor Dome via Instagram
Dome looks gleeful in the photo as she places her hand around the 28-year-old's neck.
'#tunesquad [heart emojis],' he simply captioned the post.
Rumors first started swirling about a possible romance between the two when they attended a wedding together last month looking anything but just friends.
But it wasn't until Monday when the Twilight star made it official in the form of an Instagram snap.
A day prior: Dome had shared a photo of Taylor holding her by her waist as they gleefully pose at a Halloween event
Following the announcement to his 4.7 million followers, fans wasted no time to make their opinions known on what they thought about the surprise Halloween news.
'You two have just won Halloween!!! I'm actually crying right now,' one social media user wrote in the comment section.
Another followed suit, question the Tracers star as to why he hadn't announced the news earlier.
She's no A-list: Dome is studying to become a nurse, as shown on her Instagram page
'We saw you guys at the wedding and you never announced it. Why? I've been known you guys were dating though, but congrats,' the Instagram user wrote.
On her own Instagram page, Dome took a similar approach and seemingly confirmed the romance with a sweet photo of herself and Taylor posing at a Halloween event with the same attire.
'Happy Halloweekend,' the gorgeous beauty wrote in her caption.
Dome had more or less already confirmed news of her relationship with the Michigan native after she had taken to her Instagram posting a photo of Taylor dotingly kissing her head.
Rumors: Fans had wondered whether the two were dating after the blonde beauty had posted a snap of her and Taylor at a wedding together. The actor plants a kiss on his girlfriend's forehead, making it look more than just a casual friendship.
'ITS WEDDING SZN (Not ours tho hehe),' she joked in her caption.
Before getting together with Taylor, though, Dome was already said to have been good friends with the New Moon hunk's sister Makena.
And while Taylor is no stranger to dating celebrities, having previously been linked to yet another Taylor (Swift), his new girlfriend is quite the opposite.
Dome is, surprisingly, she's a nursing student.
And since posting the doting photo that confirmed his romance with his new flame, Taylor's snap has accumulated over 530,000 likes.
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Others' Views: Lender deserves backlash
Having studied America’s mortgage meltdown and the parties who struck the match, it’s hard to resist cheering the reckoning for Countrywide Financial Corp.
Already, the nation’s largest home lender was under federal investigation for fraud, as executives allegedly understated the risks of investing in mortgage-backed securities. It was reported former CEO Angelo Mozilo — who cashed out as his company tanked — had cut special home loans for members of Congress, including U.S. Sen. Christopher J. Dodd, D-Conn. the sponsor of the Senate’s housing bailout bill.
Late last month, several states filed civil lawsuits against Countrywide and Mozilo, alleging unfair and deceptive conduct.
Of course, Countrywide’s comeuppance is nothing to celebrate, since its senior managers dragged down so much with them — employees; homebuyers; the U.S. economy.
Still, it’s refreshing to see prosecutors pursuing alleged corporate abuses with vigor.
— Peoria (Ill.) Journal Star


text:
This garden tool has a convertible handle that makes it simple to modify from towing the cart behind or pushing it ahead. This backyard cart is ideal for a range of outdoor landscapes and has a large capacity. This garden device is durable and precise, permitting you to take away your weeds without damaging your body or garden. It can be hard to seek out one of the best garden instruments on your residence, as there are such a lot of to select from.
Modern device design includes ergonomics issues, which means that they’re being designed to induce less stress on the human body when used. The most effective tools maintain the physique in a impartial place to help cut back the stress on joints and muscle tissue. An advantage of this method is it requires gardeners to exert less vitality whilst utilizing the instruments. Some trendy patents like US 7,832,125 present an instance with out hand stop provisions. In present days the gardening tools are made from mild materials and are straightforward to deal with.
Whether you’re beginning a small lawn project or overhauling your whole garden, we’ve the landscaping instruments you have to achieve the look you need. The Fiskars D-handle Square Garden Spade has a sharpened steel head that makes chopping through mud or compost extremely straightforward. This garden software additionally has an additional-massive foot pad that permits you to comfortably put more pressure into the job.
It’s essentially a handheld shovel that you’ll use for moving soil, digging, removing weeds, mixing fertilizer and far more. If you could have gardened earlier than then you know weeds can quickly grow, overtake your backyard, and kill your plants.
SHW additionally provides a good selection of excessive-quality rakes, forks, tined cultivators, and extra. The Quality and Service of the Garden Tool Company and the Schrecks are top of the hill.

domain: agronomy


The Canadian government often talks about the importance of privacy, but actions speaks louder than words. Not only has privacy reform clearly not been a priority, but the government seems more than willing to use the weak privacy rules to further other policy goals. There is an obvious price for the government’s indifference to privacy safeguards and it is paid by millions of Canadians when major privacy incidents (think Tim Horton’s or Home Depot) result in no substantive changes and no urgency for reform from the government. Indeed, as I noted yesterday on Twitter, the government has managed to rush through user content regulation in Bill C-11 and mandated payments for links in Bill C-18, but somehow privacy reform in Bill C-27 has barely moved. Some of the responsibility must surely lie with Innovation, Science and Industry Minister François-Philippe Champagne, who brings high energy to everything but privacy reform, but the decision reflects on the entire government.
The latest example occurred yesterday with the decision to ban TikTok from government devices. The official rationale came from Mona Fortier, President of the Treasury Board:
My statement announcing a ban on the use of TikTok on Government of Canada mobile devices. pic.twitter.com/X8Zfuyz5p4
— Mona Fortier 🇨🇦 (@MonaFortier) February 27, 2023
The key line – picked up by TikTok itself – is that the app is being blocked because of “concerns about the legal regime that governs the information collected from mobile devices.” That legal regime – PIPEDA – is the same regime that all Canadians rely upon to protect their privacy. It is also widely recognized to be outdated, which is why privacy reform is so badly needed. While I have my doubts about the rationale (it seems far more likely that the U.S. did it and so we followed), it is astonishing to hear a minister say that the government’s concerns with its own law is so great that it is blocking thousands of employees from installing a widely used app on their devices. If the problem lies with the law, isn’t up to the government to fix it?
Yet the lack of urgency with privacy reform extends beyond creating a two-tier security environment where government employees are subject to one standard and everyone else relies on a law the government admits is a cause for concern. Consider Bill C-18, which will be hotly debated at the Canadian Heritage committee today as MPs race to bring Google before the committee to explain why it is testing removing links to Canadian news content rather than preparing to pay hundreds of millions of dollars for those links with expectations that it fund 35% of the news expenditures of every news outlet in the country.
In the immediate aftermath of the Google report, the Canadian Association of Broadcasters sought to justify Bill C-18 on the grounds that it was about broadcasters getting their fair share of revenue from the sale of user data. That is clearly not something found in the legislation, but it suggests that broadcasters and the government are more comfortable profiting from weak privacy laws rather than addressing the core problem of the inadequate laws themselves.
In fact, Canadian Heritage Minister Pablo Rodriguez has effectively linked his digital cultural policy to the ongoing commercial success of the Internet platforms, viewing the companies as the source of funding for film and television production, music, and news. The irony that the government prioritizes “getting money from web giants” over safeguarding Canadians from potential misuse of their information by those companies should not be lost anyone. The incentive structure for Heritage is to keep the data taps flowing: more data to tech companies means more revenues which means more money for the cultural sector. Rather than seeking to regulate privacy and data governance, Rodriguez wants to profit from it by funding his policy objectives. And with an indifferent ISEd Minister and other cabinet colleagues openly admitting the law is a cause for concern but unwilling to do anything about it, Canadians may have the answer to why the government seems to embrace weak privacy rules.


text:
Creamy peonie shabby jewellery organiser
Romantic shabby chic rose with hints of cream and pink, this romantic jewellery box can hold all your gems in one place! Jewellery box comes with lid to open and bronze chain to keep it open, also one smaller pull out drawer. Plenty of space for smaller items. No holding back of different mirrors either.
Materials used: 3mm chipboard, nepalese paper, cardstock, paper flowers, bronze embellishments, bronze chain, photographed specialty paper, real mirrors, ribbons, hinge.
Measurements: 15 cm x 15cm, drawer approx 13cm x 5cm, upper space 14cm x 14cm
Please me mindful that I am made out of chipboard and paper - I don´t like wet and moist conditions, please keep water and mist away from me. If you could avoid direct sunlight for longer times that would be great - otherwise I will lose my vibrant color in time and my bronze metal parts will start to rust.


text: Great crew, great facilities, great amenities. For all you fellow road warriors, you'll feel at home here. It's a $45 cab ride to SLC, but if you like a peaceful running trail, a nearby Lifetime Fitness facility, and peace and quiet of a suburban hotel, gotta try this. THANKS! THANKS!! THANKS!! THANKS!!

domain: travel


Progress on FAA Safety Indicators Program Slow and Challenges Remain
IMTEC-92-57: Published: Aug 31, 1992. Publicly Released: Oct 14, 1992.
- Full Report:
Pursuant to a congressional request, GAO reviewed the Federal Aviation Administration's (FAA) Safety Indicators Program, focusing on: (1) the status and progress of the program; and (2) barriers to its implementation.
GAO has found that: (1) FAA has accomplished little towards its program goals; (2) development of the indicators has been delayed due to ineffective user involvement and unclear management commitment, which have also delayed development of a necessary decision support system that would integrate data from multiple sources; (3) FAA has recently redirected the program in an attempt to accelerate the identification of indicators, but less detailed indicators are being developed, which may defeat the program's goals; (4) the safety-related databases that could supply source data to the program are inaccurate, inconsistent, and often incompatible; and (5) FAA has not responded to earlier GAO recommendations that it develop a program plan that provides for effective user involvement, requisite funding, and source data integrity.


text:
Special Requirements Students selected for the clinical component of the Nursing Program must be available for daytime, evening, and occasional weekend classes.
Once blood and urine cultures are obtained starting antibiotics is the next priority. Clothing A uniform for clinical practice will be supplied but you will need to buy suitable footwear. B The Five Rights of Delegation include the right task, the right circumstances, the right person, the right direction or communication, and the right supervision or feedback.
Nursing staff should utilise their clinical judgement to determine which elements of a focussed assessment are pertinent for their patient.
The patient is not using accessory muscles to breath, however, he is using the tripod position and can only say a few words before Nursing process and clinical judgement is out of breath.
If possible, getting a lactic acid before administering fluids would be best for the patient but if blood is difficult to obtain it is more important to start the fluid boluses first.
Board staff recommends that the nurse invoking Safe Harbor keep a copy for future Nursing process and clinical judgement.
For more information see Engaging with and assessing the adolescent patient. There could be a psychological issue underlying the OD, such as depression, which would also not require the nurse to reveal anything to the Board since one of the other renewal questions asks "In the past 5 years, have you been diagnosed with or treated or hospitalized for schizophrenia or other psychotic disorder, bipolar disorder, paranoid personality disorder, antisocial personality disorder, or borderline personality disorder.
Does the infant visually fix and follow. Assess any respiratory distress. The standard that serves as the foundation for all other standards is Rule I appreciate your time. After assessment of the resident is completed and appropriate interventions are implemented, documentation of the circumstances and the assessment of the resident in the medical record are required.
The Texas Hospital Association at www. See Safe Harborabove. As your knowledge and expertise increase you will have the opportunity to move into a more senior position. When he returned home from the war at the age of 24 years of age 2 years ago he was deemed permanently disabled by both the Veterans Administration VA and the Social Security Administration.
C The registered nurse, prior to the delegation of tasks to other members of the nursing care team, evaluates the ability of staff members to perform assigned tasks for the position as based on state statutes that differentiate among the different types of nurses and unlicensed assistive personnel that are legally able to perform different tasks.
In the majority of cases, coursework such as essays; projects; dissertations is likely to be submitted on line. Chest rise and fall are symmetrical and respirations are 36 breaths per minute. Different countries also require you to take their own licensure examination.
Nurses that may be required to float to assist another unit or confronted with a potentially unsafe practice situation should be familiar with the Standards of Nursing Practice, found in Board Rule Students will be required to provide evidence of DBS clearance.
Observing the staff member during an entire shift of duty to determine whether or not the nurse has accurately and appropriately established priorities is a way to evaluate the short term abilities for establishing priorities and not assignment completion and observing the staff member during an entire shift of duty to determine whether or not the nurse has accurately and appropriately completed priority tasks is a way to evaluate the short term abilities for completing established priorities and not a complete assignment which also includes tasks that are not of the highest priority.
The message can be found at https: It helpful to utilize some of the information requested on the safe harbor form to initiate discussion surrounding the concerns about floating to areas outside of the clinical expertise and or area of competence. Must pass the interview conducted by the college admission officer.
Books Where a module specifies core texts these should generally be available on the reserve list in the library. In addition to applying to the college, students must apply to the program. Minimum grade point average, reading, writing, math, and science requirements must be met prior to applying to the RN program.
Obvious clinical signs of irreversible death include decapitation separation of head from bodydecomposition putrefactive process; decaydependent lividity dark blue staining of the dependent surface of a cadaver, resulting from blood pooling and congestiontransection, or rigor mortis body stiffness that occurs within two to four hours after death and may take 12 hours to fully develop.
If their practice placement travel costs are greater than normal daily travel, student may claim the gross amount of the costs incurred.
Entry to the nursing program is competitive and based on academic achievements. Board Position Statement Safe Harbor Peer Review is an internal process, between the nurse and the employer.
Is there a difference between the decision to initiate CPR and the decision to pronounce death. Check them out below. The library may hold copies of such texts, or alternatively you may wish to purchase your own copies. Once an IV has been established, the most important thing to do is start IV fluids, no matter if blood work was obtained or not.
Remember, delegating laboratory specimen collection is appropriate. This standard requires the nurse who is supervising other nurses to make assignments that take into account the educational preparation, knowledge, skills, physical, mental and emotional abilities of the nurses for whom the supervisor is administratively responsible.
Medical diagnosis (abbreviated Dx or D S) is the process of determining which disease or condition explains a person's symptoms and hazemagmaroc.com is most often referred to as diagnosis with the medical context being implicit.
The information required for diagnosis is typically collected from a history and physical examination of the person seeking medical care. Often, one or more diagnostic.
Clinical judgment and reasoning are essential elements of a nurse’s decision-making process and have long been considered to be the hallmarks of professional nursing (Simmons, Lanuza, Fonteyn. Higher cognitive skills are essential competencies for nurses joining the technologically and increasingly complex health care environment to provide safe and effective nursing care.
Critical Thinking, Clinical Reasoning, and Clinical Judgment Explain why knowing the nursing process is needed for clinical reasoning and passing the NCLEX ® and other standard tests. Compare and contrast the terms thinking ahead, thinking-in-action, and thinking back.
2 CHAPTER ONE What is Critical Thinking, Clinical. Clinical judgment requires clinical reasoning across time about the particular, and because of the relevance of this immediate historical unfolding, clinical reasoning can be very different from the scientific reasoning used to formulate, conduct, and assess clinical.
*BIO and BIOor the equivalent must be successfully completed within five years of entry into the first clinical nursing course.
If older than five years, the courses must be repeated.Nursing process and clinical judgement

domain: health


text:
Top 10 most ``beautiful'' clinical data in 2019
Last Update: 2020-06-19
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On the other hand, when people have a little aesthetic fatigue for PD-1 / PD-L1 or their attention has been shifted from PD-1 / PD-L1, some research and development progress and breakthroughs in other fields will be highlightedSo in 2019, we have seen the turn of over 30 years of KRAS inhibitors, the rise of RNA therapy and gene therapy, and the emergence of bispecific antibodies and ADC drugs , we are overwhelmed by hot news events, which are much more colorful than the monotony of PD-1 / PD-L1 dominating the circle of friends< br / > at the end of 2019, the magic cube of medicine screened the most "beautiful" clinical data of the year from nextpharma, taking you back to the major progress in the field of new drug research and development this yearThe so-called "Liang", on the one hand, means that these clinical research data are impressive and unforgettable; on the other hand, these clinical data can be considered as a major breakthrough in the treatment of a certain disease, the development of a certain target drug or a certain kind of drug< br / > 1 scuchiumab < br / > Company: Novartis < br / > mechanism: IL-17A monoclonal antibody < br / > disease: psoriasis < br / > research code: nct03066609 (phase III) < br / > on March 4, Novartis published a Chinese cohort data of cosentix phase III study (nct03066609) on the treatment of moderate and severe plaque psoriasis at aad2019 conference, It is the first time that the phase III study of cosentix and IL-17A monoclonal antibody for Chinese patients has appeared< br / > the study was a 52 week randomized, double-blind, placebo-controlled, international multicenter design441 Chinese patients with or without psoriatic arthritis were randomly divided into three groupsThey were given cosentix 300mg (n = 221), cosentix 150mg (n = 110) and placebo (n = 110) by subcutaneous injectionThe primary end points were PASI 75 and iga2011 0 / 1 at week 12The secondary end point was PASI 90 at week 12< br / > the results showed that cosentix had rapid effect and excellent effect in Chinese patientsThe improvement of the disease can be reflected in the third week of treatment, and the curative effect is more significant with the prolongation of treatment timeThe proportion of patients with PASI 75 was 97.7% and 80.9% of the patients reached PASI 90 at the 12th week in cosentix 300mg groupIn terms of safety, cosentyx was well tolerated, and the incidence of adverse events was consistent with previous studies< br / > the efficacy data of cosentyx for Chinese patients with psoriasis is surprisingIf compared with the previously published placebo-controlled clinical research data, the efficacy and safety of cosentyx for Chinese patients with psoriasis is still better than some international data (as shown in the table above), which is more difficult< br / > psoriasis is a chronic disease that can not be cured at presentThere are many patients in China, the number is close to 7 million, but the degree of concern is not high compared with cancerPsoriasis patients are more likely to suffer from depression, inferiority, anxiety, social fear and other psychological diseases due to the skin exposure and recurrent disease, which is also a great burden on family care< br / > however, for a long time, the first choice for psoriasis treatment in China is external medicine and phototherapy, followed by traditional medicine with large side effects such as methotrexate, cyclosporine and tretinoin, and biological preparation is usually the last choiceThe excellent data obtained by cosentix in Chinese patients and its approval for listing mark a new era of psoriasis treatment in China, which is expected to improve the treatment status of biological agents in China's psoriasis treatment and promote the progress of clinical treatment of psoriasis in China< br / > at the mid October eadv 2019 conference, the 52 week data of phase III study for Chinese patients was released by cosentixAlmost all patients who received 300mg treatment had stable effect within 52 weeks, and the proportion of patients who achieved PASI 90 and PASI 100 increased compared with the 12th week, suggesting that more patients could benefit from the long-term treatment of cosentityx< br / > 2 olapalin < br / > Company: AstraZeneca < br / > mechanism: PARP inhibitor < br / > disease: pancreatic cancer < br / > research code: Polo (phase III) < br / > on June 2, AstraZeneca announced the results of a phase III study of PARP inhibitor olapalin Code: polo at the ASCO 2019 conferenceIn polo study, 154 patients with BRCA mutation and metastatic pancreatic cancer who had not progressed after first-line platinum chemotherapy were treated with olaparide (300mg, bid) and placebo as first-line maintenance therapy until the disease progressedTo compare the efficacy and safety of olaparide and placebo in progression free survival< br / > the results showed that olaparide doubled the progression free survival time (7.4 vs 3.8 months) and the disease progression risk (HR 0.53), doubled the one-year progression free survival rate (33.74% vs 14.5%) and doubled the two-year progression free survival rate (22.1% vs 9.6%) compared with placebo< br / > the progression free survival data of polo study patients < br / > pancreatic cancer is a kind of tumor with very high malignancy and mortalityThe 5-year survival rate is only about 6% (2% - 9%), which is called "the king of cancer"Pancreatic cancer is very difficult to treatFor many years, the mainstream treatment plan is the combination of chemotherapy drugs based on gemcitabineThe improvement of survival of pancreatic cancer patients is very limitedThere is no obvious breakthrough in the past 20 years, and the clinical treatment needs are far from being met < br / > in terms of patient benefit, the BRCA mutation rate in pancreatic cancer is about 7% Polo study suggests that in patients with metastatic advanced pancreatic cancer with BRCA mutation, after obtaining valuable clinical remission with platinum containing chemotherapy, olaparide alone as the first-line maintenance treatment can significantly prolong the progression free survival period of the first-line treatment, not affect the quality of life of patients, and double the 2-year progression free survival rate, and increase the proportion of patients who can receive the second-line treatment, This is a major breakthrough in the clinical treatment of pancreatic cancer, and is of great significance for the patients with metastatic pancreatic cancer who are initially treated < br / > 3 amg-510 < br / > Company: Amgen < br / > mechanism: KRAS inhibitor < br / > disease: solid tumor < br / > research stage: phase I < br / > on June 3, Amgen orally reported the first human clinical trial results of KRAS G12C inhibitor amg-510 at ASCO 2019 Conference This open label phase I study included 35 patients with KRAS G12C mutant solid tumors who had previously received at least two lines of treatment, including 14 patients with non-small cell lung cancer, 19 patients with colorectal cancer, and 2 patients with other types of tumors According to the dosage, amg-510 was divided into 4 groups and administered 180360720960mg once a day The primary end point of the study was safety, and the secondary end points included pharmacokinetics, objective response rate (evaluated once every six weeks), response duration, and progression free survival < br / > the results showed that in 10 assessable NSCLC patients, 5 patients (50%) had partial remission (PR), 4 patients had stable disease (SD), and the disease control rate reached 90% (9 / 10); 5 patients with PR had been treated for 7.3-27.4 weeks when reading the data, and were still receiving treatment One of the patients who got PR continued to improve after reading the data and got complete remission (CR) at the 18th week In a total of 18 evaluable colorectal cancer patients, 13 achieved SD, and most received 180 or 360mg of the drug < br / > at the wclc2019 conference in September, amg-510, the data of phase I study, was further updated Among the 13 NSCLC patients whose curative effect can be evaluated, 7 patients had partial remission, 6 patients had stable disease, and the disease control rate was 100% The curative effect was equivalent to that before < br / > this phase I research result of amg-510 is of milestone significance, which makes the industry boiling Because it is the first clinical candidate drug for "non proprietary" target KRAS, we can see the solution to the problem of KRAS mutation tumor, such as pancreatic cancer with a mutation rate of 97% The incidence of KRAS G12C mutation in NSCLC is about 13%, in colorectal cancer is about 3% - 5%, in other solid tumors is about 1% - 2%, which is also a large market After amg-510, many KARs concept projects started to develop after amg-510 Up to now, nextpharma has included 22 KRAS related projects < br / > 4tetacep < br / > Company: Rongchang biology < br / > mechanism: TACI Fc fusion protein < br / > disease: systemic lupus erythematosus < br / > research stage: phase II / III < br / > on July 12, Rongchang biology announced the results of a key phase II / III clinical Study on the treatment of systemic lupus erythematosus (SLE) in the press release Hall of the Great Hall of the people In this study, 249 patients with SLE were enrolled in a multicenter, randomized, double-blind, placebo-controlled study The results showed that Sri (systemic lupus erythematosus response index) in the high-dose group was significantly higher than that in the placebo-controlled group (79.2% vs 32.0%) for 48 weeks, reaching the main end point of the study, with excellent safety performance and good patient tolerance < br / > based on the data of this study, Rongchang biology has submitted the listing application of thalacemide to nmpa, and it has been formally included in the priority review procedure by CDE This study is also one of the few highlight moments of innovative drugs in China Because the clinical trials of SLE are very difficult, many phase III studies abroad have failed < br / > lupus erythematosus is an autoimmune inflammatory connective tissue disease Systemic lupus erythematosus (SLE) is the most common (about 70%) and the most serious type of lupus erythematosus It occurs mostly in young women, and the age of onset is 20-40 years old The 10-year survival rate is less than 50% and gradually increases to 60% ~ 70% in recent years due to the involvement of kidney, respiratory and nervous system According to the epidemiological survey report in China, the prevalence of SLE is 70 / 100000, and the proportion of women is higher, reaching 113 / 100000 There are about one million patients in China, whose clinical needs are far from being met < br / > most of the traditional therapeutic drugs for SLE are non selective immunosuppressants, which are used to alleviate the inflammatory reaction, such as aspirin, cyclophosphamide, glucocorticoids, and hydroxychloroquine, which was approved by FDA in April 1955 Until March 9, 2011, beryluzumab for injection developed by GSK was approved by FDA to treat SLE It is the first new drug for lupus erythematosus approved in the world in nearly 60 years Since then, there has been no significant progress in this field < br / > baileyuzumab is a specific inhibitor of the first in class B lymphocyte stimulating factor (BLyS, also known as BAFF), which has a high affinity with soluble BLyS in serum, thus blocking the binding of BLyS to B cell receptor, inhibiting the proliferation of B cell and the differentiation of B cell to plasmocyte, so as to reduce the autoantibody produced by B cell in serum and achieve the purpose of treating SLE < br / > etaxel is a TACI Fc fusion protein, which can target BLyS / April two targets at the same time Compared with placebo, the improvement of Sri is significantly better than that of belimumab, and the price may be more people-friendly, which also makes everyone full of expectations for this domestic SLE new drug < br / > 5 dapagliptin < br / > Company: AstraZeneca < br / > mechanism: sglt-2 inhibitor < br / > disease: heart failure < br / > study code: dapa-hf study (phase III) < br / > on September 1, AstraZeneca announced the results of dapa-hf study in phase III of dapagliptin at the esc2019 conference This is the first outcome study in which sglt-2 inhibitors were used to treat heart failure in patients with reduced ejection fraction, whether or not accompanied by 2 diabetes mellitus It was found that dagglitazine can reduce the risk of major composite endpoints of cardiovascular death or heart failure deterioration by 26% The < br / > dapa-hf study analyzed the components of the primary composite endpoint separately The risk of the first deterioration of heart failure was reduced by 30% and the risk of death due to cardiovascular disease was reduced by 18% in the dagrinine group The effect of daggliptin on the main composite endpoints was similar in all key subgroups of the study < br / > the safety status of digressin in dapa-hf study
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domain: medical


Selecting the Right Data Room Technology for Your Business
If you want to conduct business online, you might be interested in info room technology. The internet supplies companies with assorted tools to manage and secure their data, including private information rooms. These tools are vital for businesses of most sizes and types, as well as the latest info room technology can help you manage your information as well as your business more effectively. Info room technology allows you to retail store, share, and process hypersensitive information more securely, and is also essential to every single successful package. However , not all data rooms are created matched. There are features and prices.
A data room facilitates businesses store and coordinate immense amounts of information. It also allows agencies to minimize the risks of secureness breaches and improve their capability to strategize for long-term development. By permitting investors watch documents without stimulating suspicions, info rooms may also help companies bring in trust and respect from investors. Choosing a reliable info room corporation means purchasing industry-leading security standards. A data room application should also contain excellent customer support to address technological issues and suggest improvements to project management. The user interface needs to be intuitive and straightforward to use. In addition , it should have zero plug-ins or other unit installation requirements, multi-lingual navigation, board portals and cellular compatibility.
Info room administrators should positively monitor access to data areas and the path both internal and external users. Using automated analytics and activity reporting is another way to track the utilization of data rooms. Young businesses are faced with a myriad of problems. In addition to dealing with potential investors, medical founders ought to find buyers and secure funding. Using investor data rooms allows them to concentrate on the larger photo, instead of the day-to-day hassles of managing details.


text:
The Arizona Attorney General’s Office Child and Family Protection Division/Child Support Services Section is seeking an Executive Staff Assistant to be the Unit Office Administrator.
This position is responsible for the office administrator functions including, providing training, guidance, and leadership to the assigned Unit. This position is responsible for supervising, organizing and assigning work to subordinate staff, selecting work methods and procedures, interviewing, hiring , training, mentoring, disciplinary actions, providing support to all staff, and attend management meetings. This position will maintain and approve attendance reports and leave request. This position oversees the functions of the support staff that are required to meet the needs of the AAGs, courts, and legal processes such as pleadings and calendar schedules.
Supervisory principles and practices
Problem solving techniques
Techniques and principles for writing briefs, opinions, motions, responses, reports and other legal documents in appropriate format
Legal concepts, terminology, principles and procedures
Ethical obligations and requirements
All applicable federal codes, state statutes, administrative rules and regulations
Methods to extract vital information from a variety of sources for legal research
File management and effective trial preparation
Extract and utilize information from statues, court decisions, legal documents and all related records
Train, plan and organize work activities for subordinate staff
Drafting complex legal documents/reports
Perform paralegal duties including but not limited to file preparation for court hearings and paternity litigation functions
Effective leadership skills
Interviewing, interacting with and obtaining information from clients, Federal, State and local government agencies, witnesses and court personnel
To identify and resolve issues
To effectively communicate orally and in writing
To work well with others
Proficiently in a variety of computer programs
Two years legal experience
• Travel may be required for this position. A valid Arizona Driver’s License is required, along with consent to have your driving record run periodically.
• All applicants under serious consideration for hire with the Attorney General's Office are required to be fingerprinted by the Office and complete a criminal background check through State and Federal agencies. A job offer cannot be tendered until the candidate has successfully passed the initial background check. Your record does not automatically constitute a bar to employment. Factors such as, but not limited to, age at time of offense and age of offense, as well as the relationship between the offense and the job for which you apply will be taken into account.
The Attorney General's Office offers a comprehensive benefits package to include:
Vacation with 10 paid holidays per year
Health and dental insurance
Life insurance and long-term disability insurance
Optional employee benefits include short-term disability insurance, deferred compensation plans, and supplemental life insurance
For a complete list of benefits provided by The State of Arizona, please visit our benefits page
• Positions in this classification participate in the Arizona State Retirement System (ASRS).
• Please note, enrollment eligibility will become effective after 27 weeks of employment.
If you have any questions, please feel free to contact Human Resources at (602) 542-8056 or email us at firstname.lastname@example.org.
During all phases of the selection process, people with a disability may request a reasonable accommodation by contacting the AG’s Human Resources Office at (602) 542-8056 or via e-mail at email@example.com. The Arizona Office of the Attorney General does not discriminate on the basis of race, color, national origin, sex, disability, religion, sexual orientation or age in its programs, activities or in its hiring and employment practices. The following division has been designated to handle inquiries regarding the non-discrimination policies: Operations Division, 2005 N. Central Ave., Phoenix, AZ 85004, Phone: (602) 542-8056, Fax: (602) 542-8000.
ARIZONA MANAGEMENT SYSTEM (AMS)
All Arizona state employees operate within the Arizona Management System (AMS), an intentional, results-driven approach for doing the work of state government whereby every employee reflects on performance, reduces waste, and commits to continuous improvement with sustainable progress. Through AMS, every state employee seeks to understand customer needs, identify problems, improve processes, and measure results.
State employees are highly engaged, collaborative and embrace a culture of public service.
The State of Arizona is an Equal Employment Opportunity Employer.

domain: law


text: Thinking about packaging for sending out the pendants I have been making (see here) I have been experimenting with making little boxes. Trying to avoid gluing or fastening I found a template for a folded box on the ever-reliable internet. Looking around for some lightweight card to experiment with, I found a pile of Christmas cards on my desk, waiting to be recycled. These proved to be a perfect weight for these little boxes. The next bit of the experiment was to decorate my own, so I printed the template onto some 180 gsm watercolour paper, splashed some coloured inks on it in a random way and folded it up. I’m not sure I will be able to use this style of box for posted items as the handles stand up and are likely to be squashed in the post, but I’m sure they will come in handy for something!

domain: design


text:
I enjoy the TV show House, largely because I like watching Hugh Laurie.
One of the small but pure joys of watching the show is watching the opening credits — or rather, listening to them. The music used in the credits comes from the song “Teardrop” by Massive Attack.
The House credits use only instrumental portions of the song. But for me, the vocal is the really stunning part. See the eerie video above to appreciate the full combination. Lyrics to “Teardrop” are here.

domain: hobby


text:
September 21, 2011
Wearing purple, about 50 library supporters gathered at the Fairfax County Government Center on Tuesday, Nov. 19, to show their support for the library board’s recommendations to the Board of Supervisors.
Stories this photo appears in:
Supervisors endorse Library Board’s recommendations to increase funding, discard beta plan.
"You can assume that the BETA Plan is dead. I will make that motion tomorrow." In her email to a concerned library patron the night before the Board of Supervisors Nov. 19 meeting, Chairman Sharon Bulova (D-at-large) put to rest speculation that the board would resuscitate the controversial beta plan aimed at streamlining the county’s library system.

domain: politics


text:
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domain: entertainment