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Interstitial osmolality is a key homeostatic variable that varies depending on the tissue microenvironment. Mammalian cells have effective mechanisms to cope with osmotic stress by engaging various adaptation responses. Hyperosmolality due to high dietary salt intake has been linked to pathological inflammatory conditions. Little is known about the mechanisms of sensing the hyperosmotic stress by the innate immune system. Here we report that caspase-1 is activated in macrophages under hypertonic conditions. Mice with high dietary salt intake display enhanced induction of Th17 response upon immunization, and this effect is abolished in caspase-1-deficient mice. Our findings identify an unknown function of the inflammasome as a sensor of hyperosmotic stress, which is crucial for the induction of inflammatory Th17 response.
Our current understanding of the mechanisms sensing stress within tissue microenvironment is limited. Much attention has been focused at cellular stress responses, where dedicated sensors, such as heat shock transcription factor 1 (HSF-1), hypoxia-inducible factor 1-alpha (HIF-1α) and nuclear factor of activated T cell 5 (NFAT5), detect different stress conditions (that is, heat shock, hypoxia and hyperosmotic stress, respectively) and mediate cell-autonomous adaptations that enable a cell to survive unfavourable environmental conditions1,2,3. However, growing evidence implicates cellular stress responses in a number of chronic inflammatory conditions, such as type-2 diabetes, atherosclerosis, inflammatory bowel diseases and neurodegenerative diseases4,5,6,7,8, suggesting a potential role of inflammation as the adaptive response to stress conditions.
Inflammation is a protective response that enables survival during infection or injury and maintains tissue homeostasis under a variety of noxious conditions9. Inflammatory responses are typically initiated by the innate immune system, when cells such as macrophages and mast cells detect noxious stimuli (for example, infection and tissue injury). Some noxious stimuli can be detected based on associated structural features, such as pathogen-associated molecular patterns10. This mode of recognition is mediated by pattern-recognition receptors, including Toll-like receptors, C-type lectin receptors, as well as cytosolic Nod-like receptors (NLRs) and retinoic acid-inducible gene 1 (RIG-I)-like helicases11. Other noxious challenges cannot be detected based on structural recognition and their detection relies instead on functional features, such as enzymatic activities, membrane pore formation and de-sequestration of cellular components. This second mode of sensing is not well characterized with the exception of NLRP3 inflammasome-mediated detection of noxious stimuli12,13,14.
The activation of caspase-1 by inflammasomes is a key signalling pathway for inflammation, which is mediated by certain NLRs, often in response to microbes15,16. Several distinct NLR-containing inflammasomes have been identified, including NLRP3 and NLRC4. Inflammasomes are molecular platforms that drive the proteolytic activation of caspase-1 and caspase-11, which results in the secretion of mature, biologically active interleukin (IL)-1β and IL-18 (refs 17, 18). Recent studies have shown that various stimuli of either biological or non-biological origin can activate the NLRP3 inflammasome, including ATP19, bacterial pore-forming toxins19,20, alum21 and several types of crystals22,23,24,25, suggesting that NLRs can sense common cellular stress events that are triggered by these diverse stimuli. Consistent with this idea, potassium (K+) efflux26, calcium (Ca2+) influx27, lysosomal membrane disruption24 and mitochondrial reactive oxygen species (ROS) production28 have been implicated as the key cellular events that induce the assembly and activation of the NLRP3 inflammasome. These cellular events, and in particular the ROS production, are often associated with diverse stress conditions. Together, these observations suggest that NLRP3 inflammasomes function to detect certain stress conditions and to translate them into inflammatory responses.
Consistent with this notion, here we identify a previously unknown function of the inflammasome as a sensor of hyperosmotic stress. We show that caspase-1 is activated in macrophages exposed to hypertonic conditions, which in turn triggers the release of mature IL-1β. This caspase-1 activation requires both NLRP3 and NLRC4 inflammasomes and is regulated by autophagy through controlling mitochondrial ROS production. We confirm our observations in vivo, showing that hyperosmotic stress induces caspase-1/NLRP3/NLRC4-dependent peritoneal inflammation. Moreover, mice with high dietary salt (NaCl) intake have higher inflammatory Th17 response upon immunization, which is abolished in caspase-1-deficient mice.
Macrophages secrete IL-1β in response to hyperosmotic stress
To investigate the potential inflammatory response to hyperosmotic stress by macrophages, addition of NaCl, sorbitol or glucose into the culture medium of cells (that is, osmotic increase) was used to generate hypertonic conditions. Bone marrow-derived macrophages (BMDMs) exposed to these hypertonic conditions, as expected, experienced acute cell shrinkage within the first minute and were able to restore near-normal cell volume by 30 min (Fig. 1a,b), demonstrating an induction of adaptation response in order to counteract the initial insult. Early-stress response genes, such as Hsph1, Ier3 and Gadd45γ, were induced in BMDMs as measured by genome-wide microarray analysis of gene expression (Fig. 1c). Moreover, a subset of genes induced by lipopolysaccharide (LPS), including Ccl2, Ccl7 and Cxcl2, were also observed in BMDMs following the exposure to the hypertonic conditions (both NaCl and sorbitol; Fig. 1c), suggesting that inflammatory signalling pathways could be also involved in the non-cell autonomous adaptation response to hyperosmotic stress. We therefore hypothesized that in addition to the cell autonomous adaptation, hyperosmotic stress in macrophages may also lead to an inflammatory response to promote tissue level adaptations.
To examine whether the inflammasome is activated by hyperosmotic stress, BMDMs were exposed to hypertonic conditions after priming with LPS to allow the expression of pro-IL-1β. There was no IL-1β or IL-6 secretion observed in non-primed BMDMs (Fig. 2a). However, after LPS priming, IL-1β secretion was induced in BMDMs incubated in hypertonic conditions with NaCl or glucose, whereas IL-6 secretion was observed in both isotonic and hypertonic conditions (Fig. 2a), suggesting that hyperosmotic stress induces the processing and release of mature IL-1β. Notably, the secretion of IL-1β by hypertonic stress was at a lower range as compared with that by the known inflammasome-activating agents such as ATP and alum (Fig. 2a). Similar results were also observed in macrophage cell line J774 (Supplementary Fig. 1). Moreover, hyperosmotic stress also induced the release of IL-1α (Fig. 2a), which, like IL-1β, has been shown to rely on caspase-1 activity29.
IL-1β has a key function in the recruitment of neutrophils, and the IL-1β-dependent peritoneal accumulation of neutrophils can be used as an in vivo assay for IL-1β production22,25,30. Using this model we found that intraperitoneal (i.p.) injection of hypertonic NaCl (0.35 M) but not isotonic NaCl (0.15 M) induced influx of neutrophils (Ly-6G+, F4/80−) and inflammatory monocytes (Ly-6G+, F4/80+low) into the peritoneal cavity (Fig. 2b). Importantly, neutrophil influx into the peritoneal cavity induced by high NaCl was largely abolished in IL-1 receptor-deficient mice (Fig. 2b,c). Together, these observations demonstrate that hypertonic stress can induce IL-1-dependent inflammatory response in vivo.
Hyperosmotic stress activates caspase-1 to release IL-1β
We next tested whether hyperosmotic stress activates caspase-1, which is necessary for processing and secretion of IL-1β. Immunoblot analysis of cell lysates from BMDMs showed that addition of NaCl, glucose or sorbitol (that is,+200 mOsm) into the culture medium induced the cleavage of caspase-1 into the active subunit p10 (Fig. 3a) and its release into the culture medium (Fig. 3b). This caspase-1 cleavage was also demonstrated in macrophage cell line J774 but not in other cell types, such as mouse embryonic fibroblasts, L929 cells and Hepa cells (Supplementary Fig. 3), suggesting that hyperosmotic stress-induced activation of caspase-1 is not ubiquitous and may be macrophage specific. Consistently, dose- and time-dependent activation of caspase-1 in BMDMs was observed specifically upon hyperosmotic challenge to BMDMs (Fig. 3c). The activation of caspase-1 was also demonstrated by using FLICA, a fluorescent inhibitor that binds covalently to the active caspase-1 (Fig. 3d). To confirm that the IL-1β secretion by hyperosmotic stress was caspase-1-dependent, caspase-1-deficient BMDMs were used. Only minimal release of IL-1β was observed as compared with BMDMs from wild-type mice (Fig. 3e). We also measured lactate dehydrogenase in the supernatants as an indicator of cell viability, which confirmed that there was no obvious cytotoxicity under hypertonic conditions (Supplementary Fig. 4). Collectively, these results suggest that caspase-1 is activated in the inflammasome complex upon hyperosmotic challenge, leading to the processing and secretion of IL-1β.
Hyperosmotic stress activates both NLRP3 and NLRC4 inflammasomes
To determine which NLR protein is involved in cell response to hypertonic stress, we used BMDMs from mice deficient in NLRP3, the adaptor molecule ASC or NLRC4. Upon hyperosmotic challenge, BMDMs lacking NLRP3, ASC or NLRC4 had minimal or reduced activation of caspase-1 as compared with BMDMs from wild-type animals (Fig. 4a), indicating that both NLRP3 and NLRC4 inflammasomes are required for caspase-1 activation. We therefore also tested BMDMs lacking both NLRP3 and NLRC4, and confirmed that further reduction of caspase-1 activation was observed in these cells (Fig. 4a). The requirement of both NLRP3 and NLRC4 in caspase-1 activation by hyperosmotic stress was further confirmed by measuring IL-1β secretion, where BMDMs lacking either of NLRP3, ASC or NLRC4 had reduced IL-1β secretion, whereas BMDMs lacking both NLRP3 and NLRC4 had minimal IL-1β secretion, similar to that by caspase-1-deficient BMDMs (Fig. 4b). In contrast, BMDMs lacking NLRP3 or NLRC4 produced minimal or normal IL-1β release, respectively, in response to ATP (Fig. 4b), the known activator for NLRP3 inflammasome19. These results suggest that NLRP3 and NLRC4 inflammasomes are responsible for sensing hyperosmotic stress in macrophages. In line with these findings, mice lacking NLRP3 and NLRC4 recruited significantly fewer neutrophils into the peritoneal cavity than wild-type mice after injection of high NaCl (Fig. 4c).
Hyperosmotic stress activates inflammasomes via mitochondrial ROS
Although the proximal mechanisms of inflammasome activation are still poorly understood, there are at least three requirements that have been suggested for NLRP3 inflammasome activation: potassium (K+) efflux, calcium (Ca2+) influx, phagolysosomal membrane rupture and ROS generation24,26,27,28. We tested these requirements in our system in order to gain insight into the mechanism involved in hyperosmotic stress-induced inflammasome activation. As expected, inhibition of K+ efflux by high extracellular KCl, and inhibition of Ca2+ influx by extracellular and intracellular Ca2+ chelators, or blocking phagocytosis by cytochalasin D inhibited inflammasome activation by ATP or alum; however, these treatments did not inhibit caspase-1 activation by hypertonic stress (Fig. 5a, Supplementary Fig. 5 and Fig. 6a), presumably because it relies on both NLRP3 and NLRC4. The response was also unaffected in macrophages lacking P2X7 receptor that could possibly sense endogenous ATP released from cells under hypertonic conditions (Supplementary Fig. 6b). In contrast, treatment of macrophages with ROS inhibitor N-acetyl-L-cysteine (NAC) blocked caspase-1 activation by hyperosmotic stress (Fig. 5a). NAC also inhibited the secretion of IL-1β (Fig. 5b), although this effect is compounded by the effect of NAC on IL-1 gene transcription31. To further confirm the ROS dependency, we used another ROS inhibitor ebselen to treat macrophages, and observed similar results (Fig. 5c). Furthermore, there was no obvious cytotoxic or off-target effect of the ROS inhibitors (Supplementary Fig. 7). Together, these results indicate that ROS is required for hyperosmotic stress-induced inflammasome activation.
We next tested whether ROS is required for both NLRP3 and NLRC4 activation. As expected, in NLRC4-deficient BMDMs, ROS inhibitors significantly suppressed IL-1β secretion (Fig. 5d), confirming that NLRP3 activation by hyperosmotic stress is mediated via ROS. Intriguingly, BMDMs lacking NLRP3 were also sensitive to the inhibition by the ROS inhibitors (Fig. 5d), suggesting that, unlike the activation by cytosolic flagellin, which is ROS independent (Supplementary Fig. 8), NLRC4 activation induced by hyperosmotic stress might also require ROS. Future study will need to address whether NLRP3 and NLRC4 are engaged into the same complex in response to ROS elevation for optimal induction of caspase-1 activation by hyperosmotic stress.
Hyperosmotic stress has been shown to increase mitochondrial ROS production32. We therefore hypothesized that mitochondrial ROS contributes to the inflammasome activation by hyperosmotic stress. To examine ROS levels in the mitochondria upon hyperosmotic challenge, we used the mitochondria-specific ROS indicator MitoSOX to selectively detect superoxide in the mitochondria of live cells. MitoSOX is targeted to mitochondria and its oxidation by superoxide leads to the generation of red fluorescence. Our data showed that MitoSOX fluorescence was enhanced by hyperosmotic challenge in BMDMs (Fig. 5e), indicating that hyperosmotic stress induces mitochondrial ROS generation. To test if the increase in mitochondrial ROS was accounted for by accumulation of dysfunctional mitochondria, we then used two types of mitochondria-specific fluorescent labels to distinguish respiring (MitoTracker Red) versus total (MitoTracker Green) mitochondria. An increase in dysfunctional mitochondria (MitoTracker Green+high, MitoTracker Red+low) was observed in BMDMs incubated in hypertonic conditions (Fig. 5f). This is in line with the loss of mitochondrial membrane potential observed by using tetramethylrhodamine methyl ester (TMRM) staining (Supplementary Fig. 9).
These data suggest that inflammasome activating ROS is generated by mitochondria. In support of this, caspase-1 activation by hyperosmotic stress was inhibited by the mitochondrial ROS inhibitor MitoTEMPO, but not by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin or the xanthine oxidase inhibitor allopurinol (Fig. 5g). Hypertonic stress-induced caspase-1 activation was also normal in BMDMs lacking gp91-phox, the subunit of NADPH oxidase (Supplementary Fig. 10). MitoTEMPO also inhibited IL-1β secretion (Fig. 5h). Combined with increased mitochondrial ROS production (Fig. 5e) and increase in dysfunctional (non-respirating) mitochondria (Fig. 5f), these data suggest that ROS produced from damaged mitochondria upon hyperosmotic stress is involved in inflammasome activation.
Autophagy is one of the major pathways by which dysfunctional or damaged mitochondria are cleared from the cells through a process known as mitophagy33. Mitophagy has been shown to control the activation of NLRP3 inflammasome by several stimuli34,35,36. Consistent with these findings, Atg5-deficient BMDMs had increased mitochondrial ROS, accumulated dysfunctional mitochondria and enhanced IL-1β secretion in response to hypertonic stress, compared with wild-type BMDMs (Fig. 6a–c). These data suggest that mitophagy plays a role in regulation of the hyperosmotic stress-induced inflammasome activation.
Collectively, the above findings have provided insights into the mechanism of inflammasome activation triggered by hyperosmotic stress. However, the upstream mechanism before inflammasome activation remained unclear. It is likely that the upstream mechanism leading to inflammasome activation couples with signalling pathways that act to sense hyperosmolarity in extracellular environments and are not macrophage-specific but common to many, if not all, cell types. We have considered various potential signalling pathways, which have been reported in different cell types previously by others, and tested a number of those. We evaluated their contribution to the inflammasome activation under hypertonic conditions by using inhibitors or BMDMs generated from knockout mice, that are specific for or lacking key signalling mediators or ion channels (Supplementary Table 1). However, none of these inhibitions or gene deficiencies in macrophages prevented the activation of inflammasome trigger by hyperosmotic stress (Supplementary Table 1 and Supplementary Figs 11–17), suggesting that the upstream mechanism leading to inflammasome activation is either redundant or mediated via an unknown pathway. Further research will be needed to clarify this aspect.
Inflammasome activation promotes Th17 response in mice on high-salt diet
High dietary salt (NaCl) intake has been associated with pathological conditions including enhanced inflammation in humans and mice. Recent studies in animals have shown that high-salt diets can lead to interstitial hypertonic salt accumulation in skin37, and promote tissue inflammation by enhancing Th17 cell differentiation in lymphoid tissues in mice with experimental autoimmune encephalomyelitis38,39. The Th17 differentiation was dependent on T-cell expression of the salt-induced serum glucocorticoid kinase 138,39. Given our findings that hypertonic stress promotes IL-1β production and the established role of IL-1β in Th17 differentiation40, we asked whether inflammasome-mediated salt-sensing mechanism in tissue compartments is involved in the induction of Th17 response in vivo. To address this, we examined whether caspase-1 is required for the Th17 polarization in mice maintained on high-salt diet. Consistent with the previous studies38,39, we observed enhanced Th17 response in high-salt diet mice immunized with ovalbumin (OVA)/LPS. Specifically, we detected increased Il17a but not Ifng mRNA expression and higher numbers of IL-17A-expressing CD4+ T cells in draining lymph nodes from wild-type mice fed high-salt diet as compared with controls (Fig. 7a,b). Furthermore, draining lymph node cells from wild-type mice on high-salt diet showed significantly enhanced IL-17A but not interferon-γ cytokine secretion upon OVA re-stimulation in vitro (Fig. 7c and Supplementary Fig. 18), indicating enhanced in vivo induction of antigen-specific Th17 cells. Most importantly, these enhancements of Th17 response by high-salt diet were abolished in caspase-1-deficient mice (Fig. 7a–c), indicating that caspase-1 activation is required for the enhanced Th17 response. These data support the idea that inflammasome activation plays an important physiological role in sensing hypertonic salt accumulation in tissue compartments and promoting Th17 responses.
The identification of a family of cytosolic innate immune sensors NLRs has led to an explosion of interest in understanding the functional role of these innate immune signalling pathways12,13,41,42. The large number of known activators of NLRP3 inflammasome, either of biological or abiotic origin, makes it highly unlikely that the NLRP3 inflammasome directly senses the stimuli that can activate it. Instead, it is now generally acknowledged that NLRP3 activation occurs mainly as a consequence of a common form of cellular stress events elicited by these stimuli. This notion is supported by recent studies where an innate immune activation through NLRP3 inflammasome can be triggered in ER-stress and oxidative-stress conditions43,44. Collectively, these observations support the notion that NLRP3 inflammasomes may function to detect cellular and tissue stress conditions and convert them into inflammatory responses. These inflammatory responses normally operate to promote defence and adaptation to stress conditions, but in chronic settings can become detrimental giving rise to a variety of inflammatory pathologies. Here we provide evidence for a inflammasome-dependent innate immune response to hyperosmotic stress and suggest that both NLRP3 and NLRC4 act to sense hyperosmolality of tissue microenvironment and mediate inflammatory responses, including neutrophil recruitment, and adaptive Th17 responses.
Our data implicating macrophages in sensing hyperosmotic stress and releasing mature IL-1β suggest that adaptation responses to physiologic hyperosmotic stress involve inflammatory components. It is in line with the observations from early studies, showing production of cytokines, including IL-1β in response to hyperosmotic stress in human peripheral blood mononuclear cells (PBMCs) and epithelial cells45,46. Here we show that this inflammatory response is triggered through the activation of caspase-1 by inflammasomes. Intriguingly, unlike the majority of known stimuli that activate the inflammasome through a specific NLR, hyperosmotic stress activates the inflammasome through both NLRP3 and NLRC4. Although the involvement of multiple NLRs has been previously reported20,47,48, it remains to be addressed how both NLRP3 and NLRC4 are engaged by a single stimulus via a pathway that involves ROS. One possibility is that NLRP3 and NLRC4 assemble into the same complex that is somehow involved in optimal induction of caspase-1 activation by hyperosmotic stress. Alternatively, there could be two distinct pathways activated by hyperosmotic stress, which are mediated via NLRP3 and NLRC4 separately and both dependent on ROS.
Caspase-1 activation through the inflammasome formation was only observed in macrophages but not other cell types such as the fibroblast and epithelial cells tested in this study. It is likely due to the fact that NLRP3 and NLRC4 are specifically expressed in macrophages. This suggests that macrophages utilize this specific array of NLRs to sense environmental hyperosmolality and mediate inflammatory responses. Moreover, this macrophage-specific sensing pathway could be uncoupled from the typical cell-autonomous stress response pathway as caspase-1 activation in macrophages was not required for the adaptation to, and survival of osmotic stress (Supplementary Fig. 4b).
Exposure of cells to hyperosmotic conditions causes numerous cellular stress events that could potentially activate the inflammasome formation, for example, cytoskeletal rearrangement due to cell shrinkage, solute influx (for example, ions, water) and ROS production. A recent study has demonstrated that, under hypo-osmotic conditions, NLRP3 inflammasome is activated through low intracellular K+ (for example, K+ efflux) that is controlled by transient receptor potential (TRP) channels such as TRP vanilloid 2 (TRPV2)49. Interestingly, here we show that inflammasome activation was affected neither by blocking K+ efflux nor by using macrophages deficient in TRPV2. Instead, the activation was inhibited by NAC and MitoTEMPO, indicating that ROS generated by mitochondria is responsible for activating the inflammasome under the hyperosmotic stress conditions. Moreover, it is regulated by autophagy through maintaining mitochondrial homeostasis (that is, mitophagy) and thus suppressing mitochondrial ROS production. Together, these findings suggest that, although both hypo-osmotic and hyper-osmotic stresses cause cell volume perturbation (cell swelling and shrinkage, respectively), they activate inflammasome formation through different mechanisms, where macrophages have evolved to employ different strategies to sense cellular perturbations in order to activate caspase-1 through inflammasomes.
The hyperosmotic stress response is critical not only to the survival of unicellular organisms, but it also has physiological functions in multicellular organisms, for example, in the kidney, where tubular epithelium is continuously exposed to extraordinarily high levels of osmolality (>1,000 mOsm)1. Cells in other tissues also deal with variations in osmolarity of their microenvironment, such as interstitial fluid. A previous study has shown that compartments in lymphoid tissues have higher osmolality relative to blood, which is maintained within tightly regulated range (for example, 285–295 mOsm in humans), and a failure in sensing hyperosmotic stress can lead to impaired adaptive immune responses50, indicating that lymphocytes-mediated immunity is contingent on adaptation to physiologic hyperosmotic stress. Also, the osmolality in tissue compartments can be affected by high dietary salt intake37. Thus, our data indicate that macrophage inflammasomes can sense hypertonic stress caused by high salt diet, promoting local inflammation and induction of Th17 response. Th17 response has been implicated in the pathogenesis of inflammation and autoimmunity in humans and mice40. Recent studies have shown that high salt diet promotes Th17 cell differentiation thereby contributing to the pathogenesis of autoimmune diseases38,39. T-cell expression of the salt-induced serum glucocorticoid kinase 1 has been shown to be important for this response38,39. Our study complements these reports by demonstrating innate sensing component for the hypertonic stress-induced Th17 response, which is likely mediated through the effect of IL-1β on Th17 cell differentiation. The findings reported here thus suggest inflammasomes and mitochondrial ROS as additional targets for Th17-mediated inflammatory diseases associated with high dietary salt intake.
Caspase-1-deficient (Casp1−/−) mice, NLRP3-deficient (Nlrp3−/−) mice, ASC-deficient (Asc−/−) mice and NLRC4-deficient (Nlrc4−/−) mice were provided by Richard Flavell (Yale University, New Haven, CT, USA). NLRP3/NLRC4-double-deficient (Nlrp3−/− x Nlrc4−/−) mice were generated in our laboratory. Atg5flox/flox mice were kindly provided by Dr Mizushima (Tokyo Medical and Dental University). Atg5flox/flox LysM-Cre+ mice were gifts of Herbert Virgin (Washington University, ST Louis, MO, USA). NKCC1-deficient (Nkcc1−/−) mice, PIP5KIβ-deficient (Pip5k1b−/−) mice, PLCβ2/PLCβ3-double-deficient (Plcb2−/− x Plcb3−/−) mice and TRPV2-deficient (Trpv2−/−) mice were kindly provided by Gary Shull (University of Cincinnati), Charles Abrams (University of Pennsylvania, Philadelphia, PA, USA), Dan Wu (Yale University, New Haven, CT, USA), Michael Caterina (Johns Hopkins University, Beltimore, MD, USA), respectively. C57BL/6 mice, IL-1R-deficient (Il1r1−/−) mice, gp91phox-deficient (Cybb−/−) mice and P2X7-deficient (P2x7−/−) mice were from the Jackson Laboratories. Mice doubly deficient in NLRP3 and NLRC4 were generated from Nlrp3−/− mice and Nlrc4−/− mice. All mice were bred and housed in accordance with National Institutes of Health- and Yale Animal Resources Center (YARC)-approved guidelines, and all studies involving mice were performed in accordance with approved Yale University Institutional Animal Care and Use Committee protocols and procedures.
Allopurinol, apocynin, ATP, C. difficile Toxin B, E. coli LPS, EIPA, glucose, incomplete Freund’s adjuvant, NAC, sorbitol, OVA, ouabain and wortmanin were from Sigma-Aldrich. Alum (Imject alum adjuvant) was from Pierce. SB203580 and JNK Inhibitor II were from Calbiochem. Hoechst, MitoTracker Green, MitoTracker Red CMXRos, MitoSOX and TMRM were from Molecular Probes. MitoTEMPO was from Enzo Life Sciences. NaCl was from American Bioanalytical. Green-fluorescent FLICA was from Immunochemistry Technologies. Flow cytometry antibodies used include Ly-6G (clone RB6-8C5), F4/80 (clone BM8) and IL-17A (clone eBio17B7), all from eBioscience.
Cells and cell treatments
Bone marrow cells isolated from the above mice strains between 6 and 12 weeks of age were grown at 37 °C in a humidified incubator in RPMI-1640 medium containing 10% FCS and 30% L929 supernatant for 7–8 days. Differentiated bone marrow-derived macrophages (BMDMs) were re-plated into 24- or 96-well plates in RPMI-1640 medium containing 10% FCS and 20% L929 supernatant 16–20 h before cell treatments. To induce hyperosmotic stress, cells were switched to hypertonic conditions made by adding a non-permeant solute such as NaCl, sorbitol or glucose into the culture medium. For an increase of 0.2 milli-osmolality (that is,+200 mOsm kg−1 H2O), 100 mM NaCl, 200 mM sorbitol or 200 mM glucose was added. Cells maintained in the regular culture medium (that is, ∼300 mOsm kg−1 H2O) were used as controls for isotonic conditions. In some experiments, LPS-primed macrophages were incubated in the presence of KCl (100 mM), or pretreated for 30 min with NAC (10 or 20 mM), ebselen (50 μM), MitoTEMPO (0.1 or 0.5 μM), apocynin (100 μM) or allopurinol (100 μM) before the stress induction.
Cell volume measurements were performed by using a Coulter Counter Model Z2 (Beckman). Cells in suspension were diluted in PBS (isotonic control) or PBS with 100 mM NaCl, 200 mM sorbitol or 200 mM glucose (hypertonic conditions;+200 mOsm kg−1 H2O) and incubated for the indicated times. Cells were analysed according to the manufacturer’s instructions. Cell sizes were also observed by differential interference contrast microscopy.
BMDMs were incubated in hypertonic conditions by adding 100 mM NaCl, or 200 mM sorbitol, or stimulated with 100 ng ml−1 LPS for 3 h and harvested into Trizol Reagent (Invitrogen). Sample preparation and hybridization to Illumina MouseWG-6 Expression BeadChip were performed at the Yale W. M. Keck facility. Gene expression heat map was created by using Cluster 3.0 and Java TreeView softwares.
BMDMs were primed with or without LPS for 3 h and incubated in hypertonic conditions as described above. Supernatants were harvested after overnight incubation. Release of proinflammatory cytokines was measured by ELISA using capture and detection antibodies against IL-6, IL-1α (BD Pharmingen), or IL-1β (eBioscience), according to the manufacturer’s protocol. LPS-primed BMDMs stimulated with ATP (5 mM) or Alum (250 μg ml−1) for 2 or 6 h, respectively, were used as positive controls for IL-1β secretion.
In vivo peritonitis
Age- and sex-matched mice of the indicated mouse strains between 6 and 8 weeks of age were injected i.p. with 0.15 or 0.35 M NaCl solution at 0.4 ml per 10 g of body weight. After 16 h, peritoneal lavage cells were stained with fluorescently conjugated monoclonal antibodies against Ly-6G (clone RB6-8C5) and F4/80 (clone BM8; eBioscience), and analysed by flow cytometry as described above. The absolute number of neutrophils (Ly-6G-positive, F4/80-negative) was determined by using CountBright counting beads (Molecular Probes) according to the manufacturer’s protocol.
Electrophoresis of proteins was performed using NuPAGE system (Invitrogen) according to the manufacturer’s protocol. Briefly, BMDMs primed with LPS for 3 h were incubated in hypertonic conditions as described above for 3 h, lysed in lysis buffer (20 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100 and 1 mM EDTA, and 1 × protease inhibitor cocktails (Roche)) and stored at −20 °C until analysed. For caspase-1 release, media supernatants were harvested after overnight incubation in hypertonic conditions as described above and stored at −20 °C until analysed. Before electrophoresis, whole-cell lysates or media supernatants were diluted with 5 × protein sample buffer (125 mM Tris-HCl (pH 6.8), 10% SDS, 50% glycerol, 0.06% bromophenol blue and 1% β-mercaptoethanol) and incubated at 72 °C for 10 min. Proteins were separated on 4–12% NuPAGE gels and transferred to polyvinylidene difluoride membrane (Millipore). To detect caspase-1, a rabbit polyclonal anti-mouse caspase-1 p10 antibody (sc-514; Santa Cruz Biotechnology) was used. LPS-primed BMDMs stimulated with ATP (5 mM) or Alum (250 μg ml−1) for 30 min or 4 h, respectively, for caspase-1 activation in whole-cell lysates, or for 2 or 6 h, respectively, for caspase-1 release in media supernatants were used as positive controls.
MitoSOX, MitoTracker Green, MitoTracker Red CMXRos and TMRM staining were performed according to the manufacturer’s instructions (Invitrogen). Data were acquired with a FACSCalibur flow cytometer (BD Biosciences) and analysed with FlowJo software (Tree Star).
High-salt diet and immunizations
Age- and sex-matched mice of the indicated mouse strains between 6 and 8 weeks of age were fed normal chow (Harlem Laboratories; TD. 96208 laboratory diet) and tap water ad libitum (control group) or sodium-rich chow (Harlem Laboratories; TD. 92034 laboratory diet) and tap water containing 1% NaCl ad libitum (high-salt group) for 4 weeks, and immunized subcutaneously in the rear footpads with 5 μg LPS and 50 μg OVA emulsified in incomplete Freund’s adjuvant. Popliteal lymph nodes were isolated from mice after 6 days later, and single-cell suspensions were made by grinding the lymph nodes between two sterile frosted slides and filtered through 70-μm cell strainer.
Total RNA was isolated from single-cell suspensions using RNeasy kit (Qiagen). Poly(A)-tailed mRNA was reverse transcribed using SMART MMLV reverse transcriptase (Clontech). PCR was performed using intron-spanning gene-specific primers and SYBR green master mix (Quanta) on Bio-Rad real-time PCR machine. The expression levels of target genes were normalized to Gapdh. Sequences of the primers used in this study are shown in the Supplementary Table 2.
Plotting of data and statistical analysis were performed using Graphpad Prism 4.0 software, and statistical significance was determined by unpaired two-tailed Student’s t-test, where P<0.05 was considered statistically significant.
How to cite this article: Ip, W. K. E. et al. Macrophages monitor tissue osmolarity and induce inflammatory response through NLRP3 and NLRC4 inflammasome activation. Nat. Commun. 6:6931 doi: 10.1038/ncomms7931 (2015).
We thank Richard Flavell (Yale University) for Casp1−/−, Nlrp3−/−, Asc−/− and Nlrc4−/− mice; Dr Mizushima for Atg5flox/flox mice; Herbert Virgin (Washington University) for Atg5flox/flox LysM-Cre+ mice; Igor Brodsky for Nlrp3−/− × Nlrc4−/− mice; Tiffany Horng (Harvard Medical School) for MitoTempo; the W. M. Keck facility at Yale for microarray analysis; Charles Annicelli and Sophie Cronin for animal care; and all members of the Medzhitov lab for insightful discussion. This work was supported by the Blavatnik Family Foundation, Else Kröner-Fresenius-Stiftung and grants from the National Institutes of Health (RO1 AI046688, DK071754, CA157461; R.M.). R.M. is an investigator of the Howard Hughes Medical Institute.
Supplementary Figures 1-18, Supplementary Tables 1-2 and Supplementary References
About this article
Nature Immunology (2016)

domain: literature


BILLINGS — Good morning.
Here comes the heat!
Daytime highs will climb back into the 90s today and we’ll be looking at well above average daytime temperatures staying with us through at least the middle of next week. Latest outlooks are keeping us warmer and drier than normal through the summer.
Conditions will stay mostly dry across the region through early next week, but a few isolated showers are possible this evening turning more widely scattered tomorrow. With the daytime heating, a few isolated thunderstorms could pop up especially Friday. These storms could produce lightning increasing the fire risk. Not much rain expected from these storms.
It will be quite hazy this afternoon as southerly winds will bring smoke up from the Big Horn/Rosebud wildfires.
Highs will be in the mid 90s today and Friday then upper 90s to low 100s across the weekend into early next week.
Nightly lows will be in the mid 60s tonight through Saturday then upper 60s Sunday into early next week.
Q2 Morning Meteorologist


MOSCOW, July 14 (RIA Novosti) - Lawyers representing Alexei Pichugin, the former head of the Yukos security service sentenced to 20 years in prison for murder, said Thursday that they were intending to go to the European Court of Human Rights.
"We will not appeal against the verdict in a higher body but will go straight to the European Court of Human Rights," Georgy Kaganer told RIA Novosti after Russia's Supreme Court upheld Pichugin's conviction at an appeal hearing.
A Moscow court jury had earlier found Pichugin guilty of murder and attempted murder, and he was sentenced to 20 years in prison.
Pichugin refused to attend today's appeal hearing. The court only read the judicial disposition, as the session was closed due to the secrecy of the case.
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Ukraine has not preserved its 1991 borders. The signing of the Geneva memorandum on April 17 reaffirmed the willingness of Russia, the United States and EU countries to reach a compromise. While the sides continue to trade tough talk and symbolic sanctions, the Kremlin and the White House are also holding a parallel dialogue on the coordinated geopolitical revision of Eastern Europe.


I haven't had any problems with actually making the adjustments to zero the scope, I just wanted to know at what range to sight it in at? Right now I have it sighted in at 25yds, but at 100yds it was impacting really high (about 7in. high). Now I understand why it does this, i'm just wandering if I were to sight it in at 50yrds if it would make it a little more accurate at the 100yd range. I'm kinda wanting to make it an accurate 25yd-200ish yd gun. So at what range should I sight in my Comp M3 Aimpoint optic? Does anyone have any info/research on ballastics for an AR-15? Thanks guys!


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Emoji Background Changer – Emoji Photo Sticker APK MOD Download 70.6
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with emoji background app for pictures 2019 you can smoothen your face easily and take acnes away from your skin would look too soft yeah, also you can narrow your nose, you can line your eyebrows as well as removing dark circles from your eyes and even brighten them, moreover you can whiten your teeth.
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domain: entertainment


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About AccuSure Fetal Doppler White Fetal Doppler
Now you can safely listen to your unborn baby's heartbeat comfortably and privately at your own home. Enjoy the amazing experience of hearing your baby's heartbeat. The Fetal Doppler is not a diagnostic device. Contact your local doctor if you have any concerns. The normal heart rate of an unborn baby is approximately 120-160bpm. The Fetal Doppler can be used above 16 weeks of pregnancy. The device brings you a clear sound of your baby's heartbeat. The probe must be contact with your skin via using gel, oil or water to obtain an accurate fetal heart rate.

domain: medical


The process of making R6M5 steel by remelting with oxygen blowing in a 20-t electric arc furnace is considered. To improve the technical-and-economic indices of the furnace operation, the following measures are tested on a series of pilot heats: the introduction of oxygen under the metal melt surface in a molten-metal bath using a consumable uncooled tube and heating of the scrap in a charging bucket by effluent gases to a temperature of 250–370°C. These measures lead to a decrease in the heat time, the metal loss, and the specific consumption of electric power and oxygen.
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A. M. Bigeev and V. A. Bigeev, Metallurgy of Steel (Izd. MGTU, Magnitogorsk, 2000).
A. N. Makarov and A. D. Svenchanskii, Optimum Thermal Conditions in Electric Arc Furnaces. Library of Electric Heat Treater (Energoatomizdat, Moscow, 1992).
V. G. Voskoboinikov, V. A. Kudrin, and A. M. Yakushev, General Metallurgy (Metallurgiya, Moscow, 2000).
O. M. Sosonkin, S. I. Gertsyk, and M. V. Shishimirov, “Method of steelmaking in an electric arc furnace and a device for its implementation,” RF Patent 2309182, Byull. Izobret., No. 30 (2007).
https://metallurgy.zp.ua/raskislenie-metalla-v-vosstanovitelnyj-period. Cited February 18, 2019.
V. A. Kudrin, Theory and Practice of Steelmaking (Mir, Moscow, 2003).
1. V. I. Yavoiskii et al., Metallurgy of Steel (Metallurgiya, Moscow, 1983).
E. V. Protopopov and L. A. Ganzer, Calculation of Steel Deoxidation and Degassing during Ladle Degassing: A Tutorial (SibGIU, Novokuznetsk, 2000).
E. F. Vegman, B. N. Zhereb’ev, A. N. Pokhvisnev, et al., Metallurgy of Steel (IKTs Akademkniga, Moscow, 2004).
A. F. Kablukovskii, Production of Steel and Ferroalloys in Electric Arc Furnaces (Metallurgiya, Moscow, 1991).
A. V. Ryabov, Material Balance Calculation: A Tutorial (Izd. YuUrGU, Chelyabinsk, 2002).
O. M. Sosonkin and V. A. Kudrin, Water-Cooled Roof of an Electric Arc Furnace (Metallurgiya, Moscow, 1985).
Translated by K. Shakhlevich
About this article
Cite this article
Gertsyk, S.I., Korolev, M.R. & Shil’nikov, E.V. Improvement of the Electric Arc Furnace Manufacture of R6M5 Steel. Russ. Metall. 2019, 1245–1249 (2019). https://doi.org/10.1134/S0036029519120085
- electric arc furnace
- metal loss
- oxygen blowing
- heating of charge


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Allegan County Resource Recovery and Hopkins Township will offer a curbside recycling program for both Hopkins Township and Hopkins Village residents.
According to Allegan County Recycling Director Ben Williams, Republic Services of Jenison was selected as the vendor for the program. Their contract begins in April and will continue for five years.
Interested households will receive a 96-gallon recycling cart with collection service on the second Wednesday of every month. Carts should be placed out the evening prior to collection day.
2013 pick-up dates will be April 10, May 8, June 12, July 10, Aug. 14, Sept. 11, Oct. 9, Nov. 13, and Dec. 11.
The following items are acceptable to place in the recycling cart: metal cans, plastic bottles or jugs with #1-7 symbols, plastic bags #2 and #4, paper, cardboard, magazines, and newspapers. Items should be clean, and cardboard should be broken down inside the cart.
All residential homes within the village/township are eligible for curbside recycling, as long as their winter property tax bill with the recycling surcharge has been paid.
The surcharge, up to $25 per year for each resident, was formerly used to fund the community drop-off site behind Bill’s Auto. That site will be closing on March 31.
According to Williams, the surcharge will not completely cover the cost of collection, so Hopkins Township has agreed to pay the difference, approximately $1,000 to $3,000 per year.
To register for curbside recycling, residents can visit www.allegancounty.org and select Recycling and Hopkins Township Sign-Up, or visit the Township Hall /Hopkins District Library where sign-up forms are available. Register by March 1. Carts will be dropped off in March.
The new plan is for residential use only. Commercial property owners may visit the county recycling website or call (269) 686-4559 to get information about their recycling options.

domain: news


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Visibility matters! Show off your pride with these charms!
A portion from each from each LGBTQ+ item sold goes towards a quarterly donation to The Trevor Project. Please visit http://donations.pocket-sushi.com for receipts and more information. All items were designed by J Tea, a queer trans man of color and the artist & owner of Pocket Sushi.

domain: artistic


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CHICAGO - A 20-year-old man has been charged with igniting a couch in a Logan Square intersection early Thursday after protesters in the neighborhood decried the lack of charges in the fatal shooting of Breonna Taylor by police.
Surveillance video allegedly shows Gavin J. Palm, of Albany Park, burning a couch shortly after midnight at an intersection in the 1900 block of North Western Avenue, according to statement from Chicago police.
As officers confronted Palm, he ran away and dropped a backpack, which allegedly held a torch lighter and an illegal amount of drugs, police said.
Officers chased him on foot and found him hiding under a chair in a backyard in the 2400 block of West McLean Avenue, police spokeswoman Kellie Bartoli said.
He was arrested and charged with two felony counts of possession of a controlled substance, and a misdemeanor count of reckless conduct for endangering the safety of motorists and pedestrians in a roadway, Bartoli said.
The charges against Palm were the only protest-related of the night, Bartoli said. Police received no reports of looting or burglary overnight connected to the protests.
Earlier Wednesday, several hundred people converged on nearby Palmer Square and Wicker Park to protest the lack of charges against the officers who fatally shot Taylor in March. Other protests were organized in Millennium Park and Auburn Gresham.
In anticipation of civil unrest, Gov. J.B. Pritzker sent in the Illinois National Guard, which could be seen Wednesday patrolling streets. The city also deployed hundreds of city trucks a barriers to commercial corridors and the Chicago Police Department headquarters, the site of another protest that evening.
Wednesday afternoon, a Kentucky grand jury brought no charges against Louisville police for the killing of Taylor during a drug raid gone wrong. Prosecutors said two officers who fired their weapons at the Black woman were justified in using force to protect themselves after they were shot at.

domain: sociology


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Location: Bowling Green, OH
Posted on: 9/3/2023
Posted by: Individual
Kane urgently needs a foster home
How long is fostering needed for?
Kayne is very loving, he’s almost a year old and loves to be loved and he is very good with other cats and dogs and kids and people in general. He’s a very good boy and he loves his stuffy’s.
Help spread the word:
- Share this page directly with someone who can foster or friends who can help
- Share on social media by clicking on the Facebook or Twitter button at the left side of this page
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domain: pet


DJ Joseph James turned his extensive knowledge of the Taylor Swift catalog into 15 minutes of seamless mashups. If you’re still thinking about the concert or looking forward to its arrival near you, it’s a great reminder of how many hits TS has put out over all her eras.
In 2006, a teenage girl from Nashville, Tennessee released her debut studio album hoping to begin a career as a country singer. Almost 17 years later, she has released 10 remarkable albums, broken numerous records, become a phenomenon in the music industry, and embarked on an ongoing tour journeying through all her eras in music called The Eras Tour. Joseph James presents Taylor Swift: The Complete Eras Megamix, producing a new musical sensation that shows a 13-minute recap of Taylor’s entire discography consisting of 231 songs from 2006 to 2023.
And if you can’t swing the concert tickets, Swift just announced that a film version is dropping at AMC Theatres on October 13.
Image: YouTube / Joseph James


Keep warm in style with these touchscreen gloves featuring a snake print and faux fur trim at the wrist. Fun and elegant- style with jeans and one of our ponchos. One Size!!Details & Care
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We are in the month of October now, and since it is Breast Cancer Awareness Month,many of us are thinking pink. We wear pink shirts, hats or bracelets, to honor breast cancer survivors and to remember those that lost their fight. But there are so many more colors in what I think of as the ” cancer rainbow.” For every type of cancer, ( and there are way too many as far as I’m concerned,), there is a color. There is light blue for prostate cancer, black for melanoma, and pink,purple,& teal for thyroid cancer, just to name a few.
No matter what type of cancer we may have been affected by, there is one common factor that brings us together: finding a cure . Cancer is an insidious disease. If not caught early, it can ravage the body, as well as the spirit. And even when it is caught early, it’s still scary to hear the words,” it’s malignant,” being directed towards you. I should know. Next month it will be 11 years since I was diagnosed with thyroid cancer.
Hearing those words about myself put fear in my heart. I was only 36 and didn’t want to die. But I was lucky. My cancer was caught early and was very treatable. I’m not the only cancer survivor in my family. My father is a 6-year survivor of prostate cancer, and in December, my mother will mark 22 years as a breast cancer survivor. Though my sister has not had caner herself( thank goodness!), she’s been affected by having a family full of survivors. I have also lost a grandfather, a cousin, and an aunt to cancer and have another cousin who is a survivor. A year ago I lost a childhood friend to breast cancer, and several years ago my best friend lost her dad to prostate cancer. Too many people have been affected by cancer one way, or another.
So let’s rally around together,and do all we can to find a cure and kick cancer in the butt!! To paraphrase that famous song from The Wizard of Oz, :
Somewhere over the rainbow there will be
A world without cancer in it for you and for me
Somewhere over the rainbow skies are blue
‘Cause we’ll do all that we can to make that dream come true !


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Member.buzz allows you to collect payments for a variety of Post Types. This contains some of the basic information on how payments are collected.
This page contains general information about the payments capabilities of Member.buzz. For information on how to manage payments in your Space, see Commerce.
Member.buzz accepts a growing number of currencies to assure access to as many customers around the world. In the table below you can find the currently accepted currencies, as well as the currencies which will be accepted in the future.
Member.buzz accepts payments through all major credit cards.
Setting up a Discount is easy at Member.buzz, which can be given to individual Members, or specific Membership types or Membership categories.
If you have any other questions regarding discounts, feel free to ask the support team at email@example.com.
A Dispute occurs when one of your customers informs their bank that their account was used to make a purchase without their consent. Whenever a dispute is filed against your Space, you will have an opportunity to respond with evidence that the customer actually did purchase your goods.

domain: finance


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Janet Burroway will read on Tuesday, February 24 at 4:30pm at the Illini Union Bookstore as part of The Carr Reading Series. This event is free and open to the public.
Janet Burroway is the author of eight novels, plays, poetry, essays, texts for dance, and children’s books. Her latest novel, Bridge of Sand appears in March from Houghton Mifflin. Her Writing Fiction : A Guide to Narrative Craft, is the most widely used creative writing text in America, and her multi-genre Imaginative Writing is in preparation for a third edition. She is Robert O. Lawton Distinguished Professor Emerita of the Florida State University.


GENERAL SEALTECH LIMITED is located in Hangzhou City and is a physical enterprise that integrates research and development, production, and sales. Our company specializes in producing Turbo Actuator Waste-gate Diaphragms,Solenoid Pulse Valve Diaphragms,Teflon / PTFE Diaphragm,Gas meter diaphragms,Rolling diaphragm,Diaphragm Valve pump Diaphragms and other products. Our company accepts various customized products and has a comprehensive ability to develop and produce special-shaped workpieces.
The company adheres to the cooperation philosophy of quality first and customer first, and has a group of long-term cooperative fixed customers in Africa, the Middle East, Europe and America, and other places. We not only have high product quality, but also excellent service.
|stainless hydraulic accumulator ptfe diaphragm
|Place of Origin
|NSF,CCS,National Sanitary Certificate,FDA,REACH,etc
|Smooth, glossy, textured, matte
|carton or according to requirements of the customers
|Online technical support
|Seal for Electrical Appliances,Machine,Valve…etc
|Heat Resistance,Abrasion Resistance,Fire Resistance…etc
|50000-200000 Piece/Pieces per Month
|Lead time (days)
|7-15 (To be negotiated)
Please note: This table is for reference only, please consult us for specific information.
stainless hydraulic accumulator ptfe diaphragm products typically have high plasticity and can be used in very low temperatures and high temperature ranges. They can also be produced using different technologies such as thin film construction, hot melt construction, and fiber construction.
If the medium used is corrosive, corrosion-resistant diaphragm materials should be selected;
Necessary protective measures should be taken to minimize damage to objects that come into contact with the already installed diaphragm.
Provide customized services exclusive to customers, and customize various diaphragm products according to their needs to meet the needs of different industries.
stainless hydraulic accumulator ptfe diaphragm—FAQs Guide
1.Can stainless hydraulic accumulator ptfe diaphragm be used in contact with food or pharmaceutical products, and what certifications are required for compliance with industry standards?
Yes, diaphragms can be used in contact with food or pharmaceutical products. Depending on the application, the diaphragm must meet certain industry standards and certifications. For food contact applications, the diaphragm must meet the requirements of the US Food and Drug Administration (FDA) and the European Union (EU) Regulation 10/2011. For pharmaceutical applications, the diaphragm must meet the requirements of the US Pharmacopeia (USP) Class VI and the European Pharmacopoeia (EP).
2.How do stainless hydraulic accumulator ptfe diaphragm maintain their flexibility and resilience over time, and what factors can lead to wear or degradation?
Diaphragms maintain their flexibility and resilience over time by being made of materials that are resistant to wear and tear. Common materials used for diaphragms include rubber, silicone, and polyurethane. These materials are designed to be durable and flexible, and can withstand a wide range of temperatures and pressures.
Factors that can lead to wear or degradation of diaphragms include exposure to extreme temperatures, exposure to chemicals, and physical damage. Additionally, diaphragms can become brittle over time due to age and exposure to the elements. To prevent wear or degradation, it is important to inspect diaphragms regularly and replace them when necessary.
3.Do stainless hydraulic accumulator ptfe diaphragm have temperature limitations, and how do they perform in extreme temperature conditions?
Diaphragms do have temperature limitations, and their performance in extreme temperature conditions can vary depending on the material used. Generally, diaphragms are designed to operate within a temperature range of -40°F to +250°F (-40°C to +121°C). In extreme temperature conditions, the diaphragm may become brittle and crack, or the material may become too soft and lose its shape. Additionally, the diaphragm may become distorted due to thermal expansion and contraction.
4.About stainless hydraulic accumulator ptfe diaphragm,What information should the customer provide to get an accurate quote from us?
Customers need to provide relevant technical requirements, drawings, pictures, industrial voltage, planned output, etc.
5.Do stainless hydraulic accumulator ptfe diaphragm have electrical conductivity properties, and can they be used in applications requiring EMI/RFI shielding?
No, diaphragms do not have electrical conductivity properties and cannot be used in applications requiring EMI/RFI shielding. Diaphragms are typically made of rubber or plastic and are used to control the flow of air or other gases.
6.Are there stainless hydraulic accumulator ptfe diaphragm designed for quick and easy replacement to minimize downtime in industrial processes?
Yes, there are diaphragms designed for quick and easy replacement to minimize downtime in industrial processes. These diaphragms are typically made of materials such as rubber, silicone, or PTFE, and are designed to be easily replaced without the need for tools or special equipment. Additionally, some diaphragms are designed with quick-connect fittings to further reduce downtime.
7.What mechanical and industrial applications can diaphragms be used in?
Diaphragms can be applied in multiple industries. GENERAL SEALTECH will introduce these to you:
1. Pumps: Diaphragms are used in pumps to separate the pump chamber from the motor, allowing the pump to move fluid without the motor becoming contaminated.
2. Valves: Diaphragms are used in valves to control the flow of liquids and gases.
3. Compressors: Diaphragms are used in compressors to create a seal between the compressor and the cylinder, allowing the compressor to compress the air or gas.
4. Actuators: Diaphragms are used in actuators to convert energy into motion.
5. Filters: Diaphragms are used in filters to separate particles from liquids and gases.
6. Seals: Diaphragms are used in seals to create a tight seal between two surfaces.
7. Pressure Vessels: Diaphragms are used in pressure vessels to contain and control the pressure of a gas or liquid.
8. Heat Exchangers: Diaphragms are used in heat exchangers to separate two fluids while allowing heat to transfer between them.
8.Are stainless hydraulic accumulator ptfe diaphragm used in safety relief valves and pressure relief devices to prevent overpressure situations?
Yes, diaphragms are commonly used in safety relief valves and pressure relief devices to prevent overpressure situations. The diaphragm is a flexible membrane that is designed to open when the pressure inside the device reaches a certain level, allowing the pressure to be released.
9.What role do stainless hydraulic accumulator ptfe diaphragm play in controlling fluid flow and preventing leaks in pumps and valves?
Diaphragms are flexible membranes that are used to control fluid flow and prevent leaks in pumps and valves. They are typically made of rubber or plastic and are designed to flex and move in response to pressure changes. When pressure is applied to one side of the diaphragm, it flexes and creates a seal that prevents fluid from leaking out. This seal is maintained until the pressure is released, allowing the diaphragm to return to its original shape and allowing the fluid to flow freely. Diaphragms are used in a variety of applications, including pumps, valves, and other fluid control systems.
10.As a stainless hydraulic accumulator ptfe diaphragm manufacturer,how can we guarantee quality?
Always a pre-production sample before mass production;Always final Inspection before shipment.
11.What types of sealing mechanisms are commonly used with stainless hydraulic accumulator ptfe diaphragm, such as clamped, bolted, or adhesive seals?
In general, stainless hydraulic accumulator ptfe diaphragm will use these sealing mechanisms:
1. Clamped Seals: Clamped seals are the most common type of diaphragm seal. They are typically used in applications where the pressure is relatively low and the temperature is not too extreme. Clamped seals are easy to install and provide a reliable seal.
2. Bolted Seals: Bolted seals are used in applications where the pressure is higher and the temperature is more extreme. They are more difficult to install than clamped seals, but provide a more reliable seal.
3. Adhesive Seals: Adhesive seals are used in applications where the pressure is low and the temperature is not too extreme. They are easy to install and provide a reliable seal. However, they are not as reliable as clamped or bolted seals.


DEFRA’s decision to hold a consultation on badger culling, in areas hit by bovine tuberculosis, must be met with a robust response from the farming industry, according to industry leaders.
“[The industry] must throw everything into persuading DEFRA to make the right decision.
This is our chance to help curb the spread of this dreadful and devastating disease said Robert Forster, National Beef Association chief executive.
“Otherwise we will be stuck with the consequences indefinitely,” said Mr Forster.
His call came after junior DEFRA minister Ben Bradshaw announced on Thursday 15 December the consultation and a range of measures to combat the disease (see News, 16 December).
Mr Bradshaw acknowledged that the badger was the primary wildlife host and that the disease had reached crisis point.
Despite introducing compulsory pre-movement testing from February 2006 he said that this alone was no longer enough.
“Evidence from around the world shows that strict cattle controls are essential if TB is to be contained and eradicated,” said Mr Bradshaw.
“But evidence also shows that it is unlikely to be successful unless, in addition, action is taken to deal with the disease in wildlife.
In this country the main wildlife vector is the badger.”
Tim Bennett, NFU president, welcomed the acknowledgement saying: “At last the government has acknowledged the need for action to tackle the reservoir of TB within the badger population in infected areas.
“Failing to tackle TB in wildlife is not compassionate.
It simply increases the risk of healthy animals contracting this debilitating and dreadful disease.”
The British Veterinary Association echoed the NFU comment.
“For too long TB control and eradication has been plagued by those who polarise its spread – laying full blame on either the cow or the badger,” said BVA president Freda Scott-Park.
“The BVA recognises that both species must be tackled if this disease is to be controlled and eradicated.”
But in stark contrast to the vets the RSPCA director of science Arthur Lindley said: “Any decision to cull badgers would be totally unacceptable to the RSPCA.
The society said it was “appalled” at the announcement and was joined by the Badger Trust in calling for more research before any cull can take place.


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Singer / Songwriter & Founder of Artist Growth
Matt Urmy is a Nashville based singer songwriter who Rolling Stone Country has named an "Artist You Need to Know." He recently celebrated the release of his new album Out of the Ashes which the Nashville Scene stated "could be the most fascinating Nashville album of the past decade...and it almost went unheard." The record was co-produced by the late (and legendary) Cowboy Jack Clement, with sessions beginning in 2010 at his home studio Cowboy Arms Hotel & Recording Spa. Within months, mixing was well under way when Clement's home and studio were lost in a fire, including the hard drives containing the nearly completed album. A year later, the raw files were miraculously recovered and Matt went to work on completing the album with the addition of the Clement inspired title track featuring John Prine. In addition to releasing a book of poetry, The Rain In The Bell, Matt is also the Founder & CEO of Artist Growth, a software company whose platform assists artists in managing their business at home and on the road. The rising company has received attention from The Wall Street Journal, Businessweek, Billboard Magazine and more and counts everyone from The Beach Boys to Earl Sweatshirt as clients. This is Matt Urmy's Fidelity High: Leonard Cohen // The Best Of // Columbia Records // 1975. Matt says, "This is one of the first LPs I ever got my hands on, and it was certainly the first one to pull me into another space and time with its songs. Something in the way they were able to create so much with such sparse production really knocked me down. The anti-gravitational and spacial aspects of the music created this frame for Leonard’s poetry, and as a viewer/listener, it was just a tremendous experience. Songs like The Partisan, Suzanne, Chelsea Hotel #2, and Bird On A Wire just blew me away. I felt I’d wandered into a portal, and when I got back there was this sense I’d been given permission, or set free in some strange way…it’s the only way I can describe the experience of being a young songwriter and falling into the multi-dimensionality of this collection of tunes."
all interview content © Fidelity High


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Health insurance: do you need it or don’t you? It’s a classic $64,000 question. If you need a hip replacement, non-urgent heart surgery, or a hysterectomy you might well wish you had insurance. If you don’t, you’ll be glad you didn’t spend the money.
Health insurance isn’t cheap. Since 2008, more Kiwis have cancelled their policies than taken out new ones. Between 2010 and 2013 the cost of health insurance for the average Kiwi household rose by nearly 30 percent. So it’s no surprise the number of people taking out insurance has been falling. Industry figures show 11,000 fewer people were insured in 2013 compared with the previous year.
Premiums also increase as you age and as new more costly treatments come into common use. When you need it most – once you’re retired – health insurance is at its most expensive.
If you get sick or suffer from a chronic condition, the public health system will help you sooner or later. It’s the “later” bit that makes health insurance worthwhile for some people. They want the peace of mind they can get treatment when they need it and not have to wait.
And the wait can be long for elective surgery – the type of surgery for which most people use health insurance.
Talk to the Ministry of Health or to government politicians and you’ll hear that the numbers of elective surgeries done each year are increasing and waiting times are reducing. The ministry says that by December 2014, it’s expected that no-one will wait longer than 4 months.
Scratch the surface of the waiting lists and picture isn’t so clear. District health boards have targets they must meet or face financial penalties. The way the DHBs deal with that is by not putting people on the waiting lists in the first place. Waiting lists show the capacity of the system, not the need.
To get on the waiting list you need to be able to show that your pain is disabling and the operation is classified as urgent. People who don’t meet the criteria won’t feature in the statistics. Access to services is also inconsistent around the country with some DHBs providing a more timely service than others.
Do you need it?
Older health insurance policies often covered day-to-day events such as trips to the GP and prescription costs. These comprehensive policies are increasingly expensive and it’s becoming more common for people to opt for elective surgery policies that will let them sidestep public hospital waiting lists.
If you’re considering taking out health insurance, you need to ask yourself:
- Can you afford to put aside savings that are earmarked just for medical bills? If the answer is yes, then you’re most likely to be better off without health insurance providing you don’t need to pay for major elective medical treatment when you’re younger.
- Can you afford to pay for doctors’ visits? Then it’s probably not worth paying higher premiums for comprehensive cover. Likewise if you can afford the premiums for budget policies that only cover GP visits and other primary healthcare such as prescriptions then you can probably afford to pay for doctors’ visits yourself.
- If you don’t make it on to a public hospital waiting list, can you afford to pay for surgery? A procedure like a knee replacement can cost between $15,000 and $28,000, according to the Health Funds Association of New Zealand. Can you afford to pay that? If a condition could stop you working, how much income could you lose?
- Are you willing to take some of the financial risk? Some companies offer cost-sharing policies that only pay a percentage of the claim. The idea is that if you have some skin in the claim you won’t want to be treated unnecessarily or have the cost of treatment balloon out of control. The other way of doing this is by taking out a higher policy excess in exchange for a lower premium.
- What are your chances of needing major surgery? Diet and exercise are vital factors in maintaining health and in reducing the risk that you’ll need medical treatment. Making lifestyle changes may do more for your health than health insurance.
We all hear stories of the person who paid $1000 in premiums and got a $50,000 operation in return. But for every one of those there will be others who paid $1000 and got nothing more than peace of mind. The fact is the health insurance company usually wins. It pays less in claims on average than the policyholder pays in premiums.
Terms and conditions
Buying health insurance can be baffling. There are many types of policies out there, ranging from those that pay for primary health care through to fully comprehensive policies that pay for almost any medical treatment.
Unlike Australia, companies here don’t have to provide policy information in a standardised format. If you do want to take out health insurance make sure you’re comparing apples with apples when looking at policies.
For example, 2 policies might each offer $200,000 cover for surgical procedures. But 1 may have more restrictions on the type of procedures available or limit the number of diagnostic tests and specialist consultations you can have.
As well as the headline cover, it’s important to read the exclusions. Is oncology (treatment for cancer) included or excluded? Some policies assume that you’ll be treated by the public health system for life-threatening illnesses such as this. However, you might prefer to be treated in a private hospital.
Another question to ask: are preventive or monitoring procedures or scans part of the policy?
Media commentator Brian Edwards and wife Judy Callingham faced this when they tried to claim for more than 1 PET (positron emission tomography) scan in a year from Southern Cross. They had to fund most of the bill for 2 of the 3 $2500 scans themselves. The couple pays $700 a month for health insurance and have had the policy for decades.
Brian Edwards said the policy didn’t make financial sense because PET scans could save Southern Cross money by identifying metastasis (spreading of the cancer) at an early stage when it could be most easily treated. “The cost is $2500 per scan. That would seem to me more economic than spending a hundred grand or more on late-stage surgery or other treatment. The PET scan is the most reliable way of telling if it has spread to other parts of the body. You can only describe this as ‘vital information’.”
As he points out, you don’t think about how many PET scans are allowed when you take out the policy. “We didn’t really know then that, 23 years later, one of us would be diagnosed with oesophageal cancer. And the other problem is that over those 23 years the terms of our policy have been changed numerous times.”
Report by Diana Clement.


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Budget builds skills for Queensland’s future
Published Tuesday, 01 December, 2020 at 04:13 PM
Minister for Employment and Small Business and Minister for Training and Skills Development
The Honourable Di Farmer
The Queensland Government has announced an investment of more than $1 billion in the 2020-21 State Budget for training and skills initiatives focussed on preparing Queenslanders for the jobs of the future.
Minister for Training and Skills Development Di Farmer said training and skills funding was vital to the government’s plan to rebuild and grow the Queensland economy.
The key investments include an additional $200 million over four years for skills initiatives and a further $21 million to extend the free TAFE for year 12 graduates and free apprenticeships for under 21s programs to young Queenslanders under 25.
“More young Queenslanders will be helped to update their skills and secure employment through an extension to the existing free apprenticeship for under 21s and free TAFE for Year 12 graduates initiatives to assist those aged up to 25 years,” Ms Farmer said.
“The extended program will apply to 165 priority qualification areas, including health services, hospitality, engineering, aged and disability support and early childhood education.”
“Young Queenslanders under the age of 25 will get access to free training in these high priority courses that we know will lead to jobs.
“More than 26,000 young people had accessed free training under our free TAFE for Year 12 graduates and free apprenticeships for under 21s programs and we want that number to grow.”
The additional $200 million investment will deliver a range of skills and training initiatives including –
- $32.4 million over two years for the TAFE Priority Skills Fund
- $25 million over two years for Pre-Apprenticeship Support
- $8 million over two years for the Social Enterprise Jobs Fund
- $5 million over two years for the First Nations Training Strategy
- And $5 million in 2020-21 for the Workforce Transition Support Program.
The Minister said the investment also included $16.51 million for the establishment of Manufacturing Skills Queensland and $100 million in TAFE facilities across the state, commencing in 2021-22 to create high quality training spaces tailored to deliver the skills local industry needs.
The projects commencing in 2021-22 include:
- $28.9 million for a Robotics and Advanced manufacturing centre at Eagle Farm TAFE
$10.6 million for a Hydrogen and Renewable Energy Training facility at Bohle TAFE
- $3.6 million for an Advanced Manufacturing Skills Lab at Bohle TAFE
- $10 million towards fitout at a new TAFE campus at Robina
- $400,000 for a business case for a proposed CQU Rockhampton campus consolidation
- $8.0 million for Stage One of the Central Queensland TAFE Centre of Excellence at CQU
- $7.5 million for the Mackay Ooralea Trade Training Centre Expansion for CQU
- $7 million for a Metal trades, manufacturing and robotics centre at Bundamba TAFE
- $3.6 million for an Advanced Manufacturing Hub at Cairns TAFE
- $4.35 million for an Agriculture and Horticulture Centre and maker space at Bundaberg TAFE
- $3.4 million for an Agricultural Centre of Excellence at Bowen TAFE
- $2.95 million for a Robotics Lab and Cyber Security Training Operation Centre at Southbank TAFE
- $4 million for Cyber Security Training Operation Centres at Mooloolaba TAFE, and Cairns TAFE
- $1.5 million for a community TAFE learning centre pilot at Yarrabilba
- $1.2 million for Nursing and Allied Health upgrades at Hervey Bay TAFE
- $1 million for Stage two of the new Rural Centre of Excellence at Toowoomba TAFE
- And $2 million for an aquaculture Training Centre at Whitsundays TAFE
“Investing in fit-for-purpose training infrastructure is essential to ensure Queenslanders can access the skills they need to secure a job and advance their careers,” Ms Farmer said.
“These projects will also support new and existing apprentices, trainees and workers in local industries.”
The successful Skilling Queenslanders for Work initiative will continue to assist another 10,000 disadvantaged people with an additional $10 million to be invested across the state in 2020-21 on top of the existing investment of $80 million.
“Skilling Queenslanders for Work continues to deliver with more than 54,500 people assisted so far and 73 per cent of participants in employment, training or a combination of both, 12 months after exiting a project,” Ms Farmer said.
“Remarkably, every dollar invested in Skilling Queenslanders for Work returns $8 for our economy.
“In addition, our Back to Work program has been extended 12 months with applications open until 30 June 2021. This extension is expected to provide businesses in regional Queensland and parts of South East Queensland with $70M worth of incentives to hire eligible jobseekers.
“This will build on the more than 23,700 people who now have jobs because of our assistance to more than 11,000 employers since July 2016.
“Importantly, more than 35 per cent of our VET investment is in areas outside of south east Queensland, supporting skills development and employment in regional areas.”
“This State Budget will support Queenslanders into training pathways in a variety of careers including traditional trades as well as growing industries in areas like digital technology and the community services sector which will continue to demand skilled workers.”
“The Queensland Government is supporting the state’s apprentices, trainees and VET students, who will be the skilled and creative workforce needed to drive economic growth and future prosperity.”
For further information visit www.desbt.qld.gov.au/training/training-careers/incentives/free-apprenticeships or www.desbt.qld.gov.au/training/training-careers/incentives/freetafe or www.desbt.qld.gov.au.
Media contact: Richard O’Leary 0447155332

domain: economics


text:
with Michelle J Lamont
In this guided meditation, I guide you through a 10-minute manifestation meditation to help you cultivate forgiveness and compassion.
We’ll start by finding a quiet, comfortable place to sit or lie down where you won’t be disrupted.
Then, we’ll take a few deep breaths and visualize a bright light surrounding us, filling us with positive energy and love. We’ll repeat powerful affirmations to release any self-judgment, resentment, or anger towards ourselves and others.
By the end of this meditation, you’ll feel fresh and energized, ready to carry the feeling of forgiveness and compassion with you throughout the day.
Listen to my latest manifestation podcast episode:
Watch Manifesting Miracles podcast episodes on my channel
Book a 15-min Free Discovery Call
Whether you need a private 1-on-1 session to understand the obstacles within your life or you have a corporate event that requires a speaker to address mindfulness in the workplace…
I am happy to accommodate you on your journey.
First consultations are free so we can begin understanding each other.

domain: psychology


text:
Butternut squash is loaded with vitamin A and vitamin C, while containing less than 100 calories and 26 carbohydrates per cup. 1 cup of this versatile winter squash also contains almost 500mg of potassium, 7 grams of fibre and about 17% of your RDA of manganese. It can help to prevent constipation and maintain a healthy digestive tract, improve eyesight, keep bones healthy and reduce inflammation.
Quinoa is among the least allergenic of the 'grains' (it actually comes from the same family as beets, spinach and chard) and has high anti-inflammatory phytonutrients. The grain contains a small amount of Omega-3 fatty acids, a higher content of mono-saturated fat than common cereal grains and is one of the most protein-rich foods in the world.
Coriander is used to treat skin inflammation, high cholesterol levels, diarrhoea, mouth ulcers, anaemia, indigestion, menstrual disorders, smallpox and conjunctivitis. This herb has eleven components of essential oils, six types of acids, minerals and vitamins.
Pomegranate seeds are low in calories and rich in fibre. They are also a good source of vitamin C and vitamin K. They are also rich in specific polyphenols, such as tannins, quercetin and anthocyanins, which may promote heart health and have anti-cancer properties. Pomegranate seeds may also reduce the risk of developing chronic diseases and aid weight management.
Manchego cheese is made in the La Mancha region of Spain from the milk of sheep from the manchega breed and is a good source of calcium, which promotes healthy bones. It contains less fat than other cheeses and is also naturally low in lactose, making it more suitable for sensitive stomaches.
Garlic was often prescribed by Hippocrates, the father of Western medicine, to treat a number of medical conditions. It has very few calories, is very nutritious and can combat sickness such as the common cold. Garlic can also reduce blood pressure, improve cholesterol, lower the risk of heart disease and improve bone health. It also contains antioxidants, which may prevent Alzheimer's disease and dementia.
Dates are rich in fibre, potassium, magnesium and copper. Health benefits include: promoting digestive health and relieving constipation; boosting heart health; weight loss; treating chronic conditions such as arthritis; preventing colon cancer; promoting a healthy pregnancy and delivery; and boosting brain health. N.B. Dates also have a high sugar content
Cayenne peppers are used therapeutically in many societies. They help to treat a variety of ailments including gout, heartburn, delirium, tremors, paralysis, fever, dyspepsia, flatulence, scarlet fever, tonsillitis and diphtheria. They are a well-known digestive aid and can also prevent migraines. Cayenne is also a circulatory stimulant, which supports detox and promotes longevity.
Brown sugar is manufactured in a different way to white sugar, which results in a different chemical structure. The process allows the sugary molasses to remain in its structure, leaving the final product in a more natural state. Brown sugar also has more flavour and texture than white sugar.
Soy sauce is low in calories and is a good source of vitamin B3, iron, phosphorous and manganese. Recent studies have suggested that it may be able to provide some digestive tract benefits Soy sauce is also, however, high in sodium and it is recommended that daily sodium intake is limited.
Sesame oil has been used in a number of ways including treating burns, preventing skin-related disorders and to promote oral health. This oil has also been shown to combat diabetes, reduce blood pressure and promote heart health.
Olive oil is known for its numerous health benefits. It is rich in mono-saturated fats and helps to lower the risk of heart disease. It contains the phytonutrient oleocanthal, which reduces inflamation and can reduce the risk of breast cancer and its reoccurrence.

domain: food


text:
5 edition of insider"s guide to sub-modalities found in the catalog.
Authors names are reversed on cover.
|Statement||Will MacDonald and Richard Bandler.|
|LC Classifications||BF637.N46 M33 1988|
|The Physical Object|
|Pagination||vii, 116 p. ;|
|Number of Pages||116|
|LC Control Number||88060928|
Johan Roelofse is on Facebook. Join Facebook to connect with Johan Roelofse and others you may know. Facebook gives people the power to share and makes. Insiders' Guides has 53 entries in the series. Borrow eBooks, audiobooks, and videos from thousands of public libraries worldwide.
The good thing with this book is that there are a lot of exercises to practice and get use to the core of submodalities. Bad point is the way it's written as most of the comment pages are transcripts from a seminar.
If you want to dive deeper in NLP, then go for it!Cited by: Insider's Guide to Submodalities book. Read 2 reviews from the world's largest community for readers.
All of the material inclded in this book is based o /5. An Insider’s Guide to Sub-Modalities. All of the material inclded in this book is based on the work of Richard Bandler. I really like the idea of this book. Unfortunately, the illustration of the authors' ideas does not work for me. It seemed that they were trying to attach too much emotional energy to a sub-modality which could then simply be re-arranged in one's mind and presto.
everything was cured/5. Richard Bandler - An Insiders Guide to Sub Modalties. An Insider's Guide to Submodalities, is a step-by-step nlp manual that enables you to use Submodalities to improve the quality of your own life and to assist others to make generative changes in their behavior.
Submodalities are the nlp component elements of representational systems: visual, auditory,kinesthetic, olfactory, and gustatory. Insider's Guide to Submodalities by Richard Bandler,available at Book Depository with free delivery worldwide/5(75). Richard is an author and co-author of numerous other books on NLP and its applications, including Frogs Into Princes (), NLP Volume I (), Tranceformations (), Reframing (), Using Your Brain (), An Insider's Guide to Sub-Modalities (), The Adventures of Anybody (), Time For a Change () and Persuasion Engineering.
An insider's guide to sub-modalities Details. Authors names are reversed on cover. Will MacDonald and Richard Bandler. Reviews. Add a review and share your thoughts with other readers. Be the first. Tags. Add tags for "An insider's guide to sub-modalities".
Be the first. An Insiders Guide To Sub Modalities Ebook or any other book is really hard, this book An Insiders Guide To Sub Modalities wont available any time so we wil ask.
Do you really want An Insiders Guide To Sub Modalities ebook?. If yes then you can proceed to download An Insiders Guide. Buy Insider's Guide to Submodalities UK ed. by Bandler, Richard, Macdonald, Will (ISBN: ) from Amazon's Book Store. Everyday low prices and free delivery on eligible orders.
Insider's Guide to Submodalities: : Bandler, /5(17). An Insiders Guide to Sub-Modalities by Richard Bandler - free mobi epub ebooks download If not, check with A and explore alternative sub-modalities to find a suitable mechanism. If they do intensify, interrupt the pattern to get rid insiders guide to sub-modalities book those unpleasant feelings.
Step 4. B instructs A to make a picture in which he sees himself as if he had. Buy a cheap copy of An Insiders Guide to Sub Modalities book by Richard Bandler. All of the material inclded in this book is based on the work of Richard Bandler.
Of Richard and his teaching, I am reminded of a passage from the Tao te Ching The Free shipping over $Cited by: Buy Insider's Guide to Submodalities by Richard Bandler, Will Macdonald from Waterstones today.
Click and Collect from your local Waterstones Book Edition: UK Ed. Get my NEW BOOK here: Check out my recommended brokers here: As the title suggests, this book is all about exploring sub-modalities.
It is a seminar transcript and includes a number of useful exercises. Motivation strategies, change personal history, belief change and, possibly most significantly, the Swish pattern are covered/5.
For more information on Submodalitites, refer to the following books: “Using Your Brain For A Change”. Richard Bandler “Change Your Mind And Keep The Change” – Steve Andreas, Connirae Andreas “An Insider’s Guide to Submodalities” – Richard Bandler and Will MacDonaldFile Size: 2MB.
If you tried the exercise, leave a comment to let me know how you got on. Also, you may want to read the follow-up article NLP: Submodalities And Your Personal Effectiveness. Recommended book: An Insider’s Guide To Submodalities by Richard Bandler and Will MacDonald.
Using Your Modalities and Submodalities Part of NLP Workbook For Dummies Cheat Sheet (UK Edition) Everything you experience is as a result of information that you take in through your senses – visual (what you see), auditory (what you hear), kinaesthetic (touch and feelings), olfactory (smell), and gustatory (taste).
Submodalities in NLP are fine distinctions or the subsets of the Modalities (Visual, Auditory, Kinesthetic, Olfactory, Gustatory, and Ad) that are part of each representational system that encode and give meaning to our experiences.
They are the building blocks of the representational systems by which we code, order and give meaning to the experiences we have. 9 UP/Library of NLP (Total 37 Books)/Bandler/Richard Bandler - Insider's Guide to 10 UP/Library of NLP (Total 37 Books)/Bandler/Richard Bandler - Patterns of the Hypnotic Techniques of Milton Erickson II [found via ].pdf.
Internet Archive BookReader PDFs and Ebooks Copy and paste one of these options to share this book elsewhere. Link to this page view Link to the book Embed a mini Book Reader 1 page 2 pages Open to this page. Finished. PDFs and Ebooks. PDFs and Ebooks ← Back to item details.
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Change Your Mind-And Keep the Change; Advanced submodalities interventions - Publisher: Moab, Utah: Real People Press. by Steve Andreas and Conniare Andreas This book is about the application of the submodalities model of NLP to personal change, counselling and coaching.Â To see this book in Amazon click here.
An Insiders Guide to Sub Modalities - Bandler ; Leaves Before the Wind: Leading Edge Applications of NLP - Grinder ; Magic in Action - Bandler ; Magic in Practice - Introducing Medical NLP - Thomson, Garner, and Khan ; Modeling With NLP - Dilts ; Practical Magic: A Translation of Basic Neuro-Linguistic Programming Into Clinical Psychotherapy.
Looking for books by Richard Bandler. See all books authored by Richard Bandler, including The Structure of Magic I: A Book About Language and Therapy (Structure of Magic), and Frogs into Princes: Neuro Linguistic Programming, and more on Instead, book a half-day tour of the island or lagoon on the first or second morning (the coolest part of the day).
The destination guide that I send to each client has a full list of activities, which can be booked once you are on island (though any private tours tend to sell out in advance).
Now comes the part of “How do you find your sub-modalities?” You can always relive the past expe. To order a book or make an enquiry please email the Librarian by clicking here.
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Price New Immunosuppressive Modalities and Anti-Rejection Approaches in Organ Transplantation (Medical Intelligence Unit) Jerzy W. Kupiec-Weglinski For Kindle. Richard Bandler - Insider's Guide to : MB: Richard Bandler - Patterns of the Hypnotic Techniques of Milton Erickson II [found via MB: Richard Bandler - Time For A Change (OCR) v [found via ].pdf: KB: Richard Bandler - Using Your : MB.
Insiders’ Guide to Montreal this tiny English-language book shop packs a surprisingly robust selection of titles by local authors and others you won’t find at most large s: Just make sure to book early.
Speaking of making early plans, the attractions in Florence are very popular and it’s typically best to buy your tickets ahead of time. Purchase tickets for the Uffizi Gallery online, and get a combined pass for the landmarks in Piazza del Duomo (the Cathedral and Duomo, Giotto’s Campanile, and the Baptistery).Author: Passion Passport.
Books shelved as nlp: Frogs Into Princes by Richard Bandler, The Structure of Magic I: A Book about Language and Therapy by Richard Bandler, NLP: The New.
Read our Telegraph Travel expert guide to Los Angeles, including the best places to stay, eat, and drink as well as the top things to do, and all of the information that you need to know before Author: Luciana Bellini.Sample for: Insider's Guide to Academic Writing: A Rhetoric and Reader Summary Praised for its accessible approach to teaching disciplinary writing, the first edition of An Insider's Guide to Academic Writing was embraced by instructors and students at two-year and four-year schools Edition: 2nd

domain: library


text: Art Corpuz started in Stained Glass in 1994 and concentrated in the Tiffany style of stained glass construction. The incorporation of lead channel techniques and stained glass panel repairs opens an avenue of challenges inherent with each piece. He develops and constructs customized panels. He enjoys the challenge of new designs, patterns and techniques, which engages the creative process and making each panel unique. Art is pursuing other avenues of glass-works to include lamp-working, beading and fusing techniques.


text:
Good news on Kashmir soon: GilaniBy ANI
Sunday, October 17, 2010
LAHORE - Pakistan Prime Minister Yousaf Raza Gilani has congratulated his nation and the people of Kashmir on ‘international recognition’ of Pakistan’s demands on the issue, and said that people would hear good news soon.
During a meeting with AJK Prime Minister Sardar Attique Ahmed Khan, Gilani reiterated that Pakistan is committed to resolving the Kashmir issue, and it would not compromise on its principles regarding the Kashmir policy, the Daily times reported.
“Recognition by the international community of this long-standing demand of Pakistan for resolution of the Kashmir issue augurs well for the cause and Kashmiris will soon receive good news in this regard,” he said.
Attique briefed Gilani on his participation in high-level meetings in Brussels for resolution of the Kashmir issue, as well as his input at the United Nations, European Union and the Organisation of Islamic Conference meeting.
Adoption of the Kashmir dispute as an international issue by the European Union Parliament after 63 years was the Pakistan government’s greatest achievement on the foreign affairs front, Attique said, adding that the EU had started a hearing in its parliament for resolution of the long-standing issue.
He also said that the UN, EU and OIC had included the Kashmir issue in their official businesses after adopting a common mechanism for its resolution, adding that this success was an outcome of a dedicated struggle of the present Pakistani government based on a principled stance. (ANI)

domain: politics