Generic lasix online

People across NSW who have received both doses of a hypertension medications treatment will be allowed more freedoms next month after NSW hit the target of six million jabs.This is the first step in the roadmap and further generic lasix online freedoms will follow for those who have had the jab when the state hits new vaccination targets of 70 and 80 per cent. Following consultation with Dr Kerry Chant and her team, as well as the NSW Chief Psychiatrist Dr Murray Wright, the following individual freedoms will be allowed generic lasix online for adults who have received both doses of the hypertension medications treatment.From 12.01am, Monday, 13 September:For those who live outside the LGAs of concern, outdoor gatherings of up to five people (including children, all adults must be vaccinated) will be allowed in a person’s LGA or within 5km of home.For those who live in the LGAs of concern households with all adults vaccinated will be able to gather outdoors for recreation (including picnics) within the existing rules (for one hour only, outside curfew hours and within 5km of home). This is in addition to the one hour allowed for generic lasix online exercise.

Premier Gladys Berejiklian thanked the millions of people across NSW who came forward to receive their treatment, helping hit the six million doses target.“We are so grateful for every person who comes forward to get vaccinated because the more jabs we get into arms, the sooner we can lift restrictions,” Ms Berejiklian said.“We appreciate the community’s patience in the lead up to 13 September, this additional time will allow the recent surge of treatments to take effect.”As part of the roadmap when the following targets are hit, freedoms will be generic lasix online as follows:70 per cent full vaccination. A range of family, industry, community and economic restrictions to be lifted for generic lasix online those who are vaccinated.80 per cent full vaccination. Further easing of restrictions on industry, community and the economy.The government is also investigating trials of certain industries in coming months, as a proof-of-concept measure to prepare the businesses to open up and operate in a hypertension medications-safe way.Deputy Premier John Barilaro said this roadmap is our path to freedom and is our biggest incentive yet to get vaccinated so we can return to a level of normality.

€œThe roadmap announced today outlines a clear pathway forward in which generic lasix online a range of family, industry, community and economic restrictions will be lifted for those that are fully vaccinated when NSW hits 70 per cent,” Mr Barilaro said. €œHaving a meal with loved ones, or having a drink with friends is just around the corner, but generic lasix online to get there, we need to keep up momentum in the vaccination rollout.” Health Minister Brad Hazzard said two doses of the treatment not only helps protect people from hospitalisation and death, but also helps reduce transmission.“Two treatment doses leads to around a 90 per cent overall reduction in transmission of the lasix,” Mr Hazzard.If you are not booked in for a hypertension medications treatment, please book an appointment as soon possible.There are several options to receive your ‘proof of hypertension medications vaccination’:Download your hypertension medications digital certificate via the Express Plus Medicare mobile app or your Medicare online account through myGov. You can add your generic lasix online hypertension medications digital certificate to your Apple Wallet or Google Pay.Instructions are available on the Services Australia website.

If you can’t get generic lasix online proof online, your vaccination provider can print your immunisation history statement for you.Call the Australian Immunisation Register on 1800 653 809 (Monday to Friday 8am to 5pm) and ask for your statement to be sent to you. It can take up to 14 days to arrive in the post.If you’re not eligible for Medicare you can call the Australian Immunisation Register and request your certificate be mailed to you or add your hypertension medications certificate to your digital wallet using the Individual Healthcare Identifiers service (IHI service) through myGov.For the latest information visit the NSW Government website..

Lasix and water intake

Activating mutations lasix and water intake in receptor guanylyl cyclase C (GC-C), the target of gastrointestinal peptide hormones guanylin and uroguanylin, and bacterial heat-stable enterotoxins cause early-onset diarrhea and chronic inflammatory bowel disease https://www.kampradmedia.de/kamagra-oral-jelly-100mg-factory-discount-prices/ (IBD). GC-C regulates ion and fluid secretion in the gut via cGMP production and activation of cGMP-dependent protein kinase II. We characterize a lasix and water intake novel mouse model harboring an activating mutation in Gucy2c equivalent to that seen in an affected Norwegian family. Mutant mice demonstrated elevated intestinal cGMP levels and enhanced fecal water and sodium content. Basal and linaclotide-mediated small intestinal transit was higher in mutant mice, lasix and water intake and they were more susceptible to DSS-induced colitis.

Fecal microbiome and gene expression analyses of colonic tissue revealed dysbiosis, up-regulation of IFN-stimulated genes, and misregulation of genes associated with human IBD and animal models of colitis. This novel mouse model thus provides molecular insights into the multiple roles of intestinal epithelial cell cGMP, which culminate in dysbiosis and the induction of inflammation in the gut. Monogenic intestinal epithelium defects contributing to pediatric lasix and water intake inflammatory bowel disease (IBD) have been described and are not readily amenable to current treatment regimens (Leung and Muise, 2018. Nambu and Muise, 2021). Among the genes associated with very early–onset IBD are mutations in the receptor guanylyl lasix and water intake cyclase C gene (GUCY2C.

Bose et al., 2020. Crowley et al., 2020). The receptor encoded by this gene, lasix and water intake guanylyl cyclase C (GC-C), is the target of the gastrointestinal hormones guanylin (encoded by GUCA2A) and uroguanylin (encoded by GUCA2B. Arshad and Visweswariah, 2012. Basu et lasix and water intake al., 2010).

GC-C is predominantly expressed along the gastrointestinal tract, where it regulates fluid and ion transport across the intestinal epithelium (Waldman and Camilleri, 2018). Ligand binding to GC-C results in elevated 3′5′-cyclic guanosine monophosphate (cGMP) levels in the intestinal cell and activation of cGMP-dependent protein kinase II (PKGII. Lohmann et al., lasix and water intake 1997). PKGII phosphorylates the cystic fibrosis transmembrane conductance regulator (CFTR) and the sodium-hydrogen exchanger, NHE3 (Chen et al., 2015. Golin-Bisello et al., lasix and water intake 2005).

Phosphorylation of CFTR increases secretion of chloride and bicarbonate ions, while phosphorylation of NHE3 inhibits sodium uptake by the intestinal epithelial cell (IEC. Chen et al., 2015). The ensuing osmotic imbalance across the IEC causes efflux of water from the cell required for lasix and water intake mucus hydration and passage of the bolus of food along the gut (Arshad and Visweswariah, 2013). Familial GUCY2C diarrhea syndrome (FGDS) was first described in a Norwegian family where >30 individuals reported diarrhea of varying severity from infancy onward (Fiskerstrand et al., 2012). The autosomal dominant mutation mapped to GUCY2C resulted in lasix and water intake a change of Ser840 to isoleucine, present in the guanylyl cyclase domain.

The mutation resulted in hyperactivation of GC-C whereby the mutant receptor elicited greater levels of cGMP on stimulation with ligands. These elevated cGMP levels presumably overactivated downstream signaling, resulting in increased fluid and ion secretion and diarrhea (Fiskerstrand et al., 2012). Subsequently, we characterized an additional four activating mutations in unrelated children in Europe, who also presented with severe and debilitating diarrhea, lasix and water intake detectable in utero as a greatly distended abdomen in the fetus (Müller et al., 2016). The mutations (Lys507Glu, Leu775Pro, Arg792Ser, and Asn850Asp) were present in different domains of the receptor, including the kinase-homology domain, the linker region, and the catalytic domain (Bose et al., 2020. Mishra et al., 2018) lasix and water intake.

Patients suffering from FGDS and children with de novo mutations in GUCY2C present with Crohn’s disease (CD)–like symptoms and colitis in addition to diarrhea (Fiskerstrand et al., 2012. Müller et al., lasix and water intake 2016). GC-C is the target of bacterial heat-stable enterotoxins (STs) produced by enterotoxigenic Escherichia coli, one of the major causes of watery diarrhea in developing countries (Schulz et al., 1990). The Food and Drug Administration–approved drugs linaclotide and plecanatide, which are used to treat constipation, comprise the cysteine-rich core sequence of ST or uroguanylin, respectively, and activate GC-C to induce cGMP production (Shah et al., 2018). However, no drugs are available to alleviate diarrhea lasix and water intake mediated by GC-C, though molecules that may inhibit proteins downstream of GC-C show promise (Bijvelds et al., 2018).

Knockout mice for GC-C have been studied for several years (Schulz et al., 1997). While they show no lasix and water intake apparent signs of constipation, reports indicate that they demonstrate neurological symptoms (Brierley, 2012. Mann et al., 2019), and suppression of uroguanylin-mediated GC-C signaling in mice results in obesity (Valentino et al., 2011). We have demonstrated that GC-C−/− mice are more susceptible to oral Salmonella enterica enterica serovar Typhimurium (Majumdar et al., 2018). Inactivating mutations have been reported in GUCY2C, lasix and water intake with infants presenting with meconium ileus at birth.

These children were the outcome of consanguinity, with mutations present in a homozygous state (Romi et al., 2012. Smith et al., lasix and water intake 2015. Woods et al., 2019). The lack of models for diarrheal disease mediated by hyperactive GC-C to investigate the link between GC-C, cGMP, and gut inflammation prompted us to develop a mutant mouse harboring a mutation in Gucy2c, equivalent to the S840I mutation found in the Norwegian family. Here, we validate this model in terms of increased frequency of passing watery feces, regulation of Cftr and Nhe3 lasix and water intake activity in vivo, and enhanced susceptibility of these mice to colitis.

Analysis of the fecal microbiome in mutant mice and global colonic gene expression provided the underlying explanation for inflammation observed in patients harboring activating mutations of GC-C. Therefore, this mutant mouse serves as a preclinical model for GUCY2C-mediated lasix and water intake secretory diarrhea associated with IBD and the more prevalent infectious diarrheal illness caused by GC-C activation. Transcript levels of Gucy2c and uroguanylin (Guca2b) were similar in the ileum and colon of wild type and S839I mice, while levels of guanylin (Guca2a) were significantly lower in S839I mice (Fig. 2 A). The gut is the major site of synthesis lasix and water intake of guanylin and uroguanylin.

Therefore, serum levels of the propeptides are reflective of levels produced in the gut. However, we did not see lasix and water intake a significant decrease in propeptide hormone levels in S839I mice sera (Fig. S3). Western blotting revealed that GC-C expression was similar in wild type and S839I mice (Fig. 2 B), as was ST binding to membranes prepared from epithelial lasix and water intake cells (Fig.

2 C). The presence of GC-C harboring hyperactivating mutations in patients’ gut was hypothesized to lasix and water intake result in elevated intra-epithelial cell cGMP in response to guanylin and/or uroguanylin (Fiskerstrand et al., 2012. Müller et al., 2016). As shown in Fig. 2 D, steady-state lasix and water intake levels of cGMP in epithelial cells were ∼5- to 10-fold higher in S839I mice.

Therefore, mutant mouse GC-C indeed elicited a greater response in terms of cGMP production in response to the endogenous ligands. Patients with FGDS demonstrated a delayed gut transit time due to regurgitation of gut contents in the small intestine (von Volkmann et al., 2017, 2016) lasix and water intake. We estimated gut transit in wild type and S839I mice but observed no change in S839I mice (Fig. 2 E) lasix and water intake. Patients with activating mutations in GUCY2C pass multiple, watery stools (Fiskerstrand et al., 2012).

We monitored the number of fecal pellets passed by mice in 10 min and observed that S839I mice passed a greater number of fecal pellets (Fig. 2 F) lasix and water intake. The water content in feces produced by S839I mice was higher (Fig. 2 G) lasix and water intake. These phenotypes mirror the symptoms of diarrhea seen in FGDS, suggesting that S839I mice can be used to understand processes that regulate the frequent episodes of bowel evacuation seen in FGDS patients.

Elevated cGMP levels in IECs stimulate enhanced chloride and bicarbonate secretion through CFTR following phosphorylation by cGMP-dependent protein kinase G II (Fig. 3 A lasix and water intake. Golin-Bisello et al., 2005. Lin et al., 1992) lasix and water intake. In addition, inhibitory phosphorylation of the sodium-hydrogen exchanger NHE3 (SLC9A3) results in reduced sodium ion import into cells and consequent increase in luminal and fecal sodium (Chen et al., 2015).

Transcript levels of PrkgII were reduced in the colon of S839I mice as was the level of the protein (Fig. S3 B), suggesting that some effects of cGMP in the colon could be PKGII lasix and water intake independent. While Cftr transcripts were similar in both wild type and S839I mice, levels of Nhe3 were lower in both the ileum and colon in S839I mice (Fig. 3 B) lasix and water intake. We measured small intestinal transit rates in wild type and S839I mice.

Gastric emptying (GE) was slightly enhanced in wild type mice following treatment with the Cftr inhibitor, but not significantly, though GE is reported to be enhanced in CFTR patients (Collins et al., 1997). No change in GE was seen in S839I lasix and water intake mice (Fig. 3 C, upper panel) either in the presence or absence of a Cftr inhibitor. However, the lasix and water intake extent of migration of the dye in the small intestine, as measured by the geometric center (GC), was significantly higher in S839I mice (Fig. 3 C, lower panel) treated with vehicle alone.

On administration of the Cftr inhibitor, an increase in GC in wild type mice was seen (Fig. 3 C, lower panel), which agrees with the paradoxical observation that upper small intestinal transit is increased in cystic fibrosis patients lasix and water intake (Hedsund et al., 2012). Importantly, a dramatic reduction in the GC was seen in S839I mice treated with the Cftr inhibitor (Fig. 3 C, lower panel), demonstrating that lasix and water intake the increased basal transit rate in S839I mice was almost solely due to CFTR activation. We then administered linaclotide to more potently activate GC-C (Bryant et al., 2010).

GE was lasix and water intake higher in S839I mice (Fig. 3 D, upper panel) when mice were gavaged with buffer alone, in contrast to what was seen in mice gavaged with the vehicle used to dissolve the Cftr inhibitor (Fig. 3 C, upper panel). GC-C and uroguanylin expression has been reported in the stomach lasix and water intake of mammals, albeit at low levels (Date et al., 1999. London et al., 1997), and the increased GE could be a consequence of hyperactive GC-C in the stomach.

On linaclotide treatment, the GC was increased in lasix and water intake both wild type and S839I mice (Fig. 3 D, lower panel). However, transit was significantly higher in S839I mice. Therefore, ligand-mediated activation of hyperactive GC-C causes more rapid migration down the small lasix and water intake intestine. We had observed a transcriptional down-regulation of Nhe3 in both the ileum and the colon of S839I mice in comparison with wild type mice (Fig.

3 B) lasix and water intake. We monitored expression of Nhe3 and saw a significant reduction in protein expression in both the ileum and colon of S839I mice (Fig. 3 E). Increased bicarbonate efflux from the cell, lasix and water intake due to elevated cGMP levels and Cftr activity, along with reduced expression of Nhe3 should increase sodium levels and luminal pH along the gut. In agreement with this, luminal pH in the ileum of S839I mice was higher than in wild type mice (Fig.

3 F) lasix and water intake. However, a significant increase in luminal sodium was observed only in the colon (Fig. 3 G) and was correlated with higher fecal sodium content (Fig. 3 H) lasix and water intake. This suggests that Nhe3 may not be the main contributor to sodium import in the small intestine.

However, down-regulation of Nhe3 coupled with lasix and water intake inhibition of its activity due to elevated cGMP levels in the colon manifests in enhanced excretion of fecal sodium, as seen in children with hyperactivating mutations in GC-C that show congenital sodium secretory diarrhea (Müller et al., 2016). We evaluated the extent to which Nhe3 is inhibited by elevated cGMP levels in the gut in S839I mice by administering tenapanor, a specific Nhe3 inhibitor. While GE was again higher in S839I mice administered vehicle alone (Fig. 3 I, upper panel), no further lasix and water intake increase was seen in both wild type or S839I mice following administration of tenapanor. However, in agreement with earlier studies (McHugh et al., 2018), administration of the inhibitor to wild type mice increased the GC (Fig.

3 I, lower lasix and water intake panel). In S839I mice, the already elevated basal transit was further enhanced by tenapanor treatment (Fig. 3 I). We attribute lasix and water intake this enhanced increase in small intestinal transit to the prevalent lower levels of Nhe3 present in S839I mice and efficient inhibition by tenapanor. Patients harboring activating mutations in GUCY2C present with varying degrees of inflammation in the gut (such as esophagitis, irritable bowel syndrome [IBS], CD, and ulcerative colitis [UC].

Fiskerstrand et lasix and water intake al., 2012. Müller et al., 2016). Administration of DSS to mice causes lasix and water intake death of epithelial cells and compromises barrier function (Wirtz et al., 2017). We administered DSS to mice and monitored weight loss and damage to the colon. S839I mice were more susceptible to DSS as evidenced by greater weight loss (Fig.

4 A, left panel) and higher lasix and water intake disease activity index (Fig. 4 A, right panel). Colonic shortening was observed in both wild type and S839I mice after lasix and water intake DSS treatment (Fig. 4 B). Transcript levels of GC-C and guanylin are reduced in biopsies taken from human UC and CD patients (Brenna et al., 2015.

Lan et lasix and water intake al., 2016). Following the induction of colitis by DSS, transcript levels of Gucy2c and Guca2a were reduced in both wild type and S839I mice (Fig. 4 C) lasix and water intake. Histological evaluation of the colon in wild type and S839I mice revealed no difference in colon architecture or changes in crypt depth, indicating that IEC turnover was similar in S839I mice (Fig. S3 C).

After DSS treatment, a greater lasix and water intake degree of crypt abscesses and destruction of colonic mucosa was observed in S839I mice (Fig. 4 D). Concomitant with greater lasix and water intake mucosal damage, fecal lipocalin, a sensitive marker for inflammation in the gut (Chassaing et al., 2012), was increased in S839I mice (Fig. 4 E). Taken together, our results show that S839I mice harboring an activating mutation in Gucy2c reveal roles of cGMP in regulating gut function and enhanced colonic susceptibility to damage in a colitis model.

Notably, GC-C knockout mice are lasix and water intake resistant to DSS-induced colitis (Steinbrecher et al., 2011). To explore global changes seen in the gut because of the presence of hyperactive GC-C, we took unbiased approaches by characterizing the fecal microbiome and the transcriptome in colonic tissue. The altered lasix and water intake pH and sodium ion concentrations in the gut lumen may result in dysbiosis that could predispose to colitis. We therefore performed 16S ribosomal RNA (rRNA) gene amplicon sequencing of fecal samples collected from wild type and S839I mice. Unconstrained ordination through principal component analysis with Bray-Curtis dissimilarity metrics displayed a clear separation between the two sets of mice (analysis of similarities P value = 0.001.

Fig. 5 A). There was a reproducible trend of reduced α diversity (P <. 0.1) in the fecal microbiome of S839I mice (Fig. 5 B), and relative abundance plots demonstrated differences at the phylum- and genus-levels (Fig.

5 C). An increased abundance of potential opportunistic pathogens (Anaeroplasma, Desulfovibrio, Mucispirillum, and Paraprevotella) was seen in S839I (Fig. 5 D). Members of the genus Paraprevotella are associated with colonic CD and produce succinic acid, increased levels of which are reported to be associated with microbiome dysbiosis and intestinal inflammation in patients with IBD and animal models of chronic colitis (Macias-Ceja et al., 2019. Walters et al., 2014).

The genus Mucispirillum is also significantly higher in S839I mice (Fig. 5 D). Exposure of Nod2−/−Cybb−/− C57BL/6 mice to a mucus-dwelling Gram-negative pathobiont of rodents, Mucispirillum schaedleri, has been reported to trigger the development of CD-like colitis (Caruso et al., 2019). Desulfovibrio was significantly enriched in S839I mice as seen in the colonic mucosal and fecal microbiome of UC and CD patients (Rowan et al., 2010). Levels of protective bacteria such as Colidextribacter, Dorea (short-chain fatty acid producers), Dubosiella, and Lactobacillus (possessing anti-inflammatory properties) were significantly reduced in S839I mice (Fig.

5 E). Colidextribacter and Dorea belong to Clostridiales cluster IV and Clostridium cluster XIVa in the phylum Firmicutes, respectively. These taxa are known to produce short-chain fatty acid and are reported to be less abundant in the ileal biopsy and fecal samples isolated from CD patients (Nagao-Kitamoto and Kamada, 2017). Lactobacillus strains restore the commensals and gut homeostasis in intestinal disorders (Blaser, 2014). Therefore, the lower abundance of these genera in S839I mice suggests that these animals may be more susceptible to environment-induced colitis and inflammation in the gut, as reported in FGDS patients (Fiskerstrand et al., 2012).

Analysis of predicted functional consequences of the variation in abundance of taxa between the mice (Narayan et al., 2020) indicated significant differences in 28 KEGG pathways (Table S3). Those linked to host immunity were enriched in S839I mice and included NOD-like receptor signaling, antigen processing and presentation, IL17 signaling, and Th17 cell differentiation (Fig. 5 F). KEGG pathways for bacterial chemotaxis and flagellar assembly, both associated with cell motility in the microbiome, were significantly higher in S839I mice (Table S3). Pathways that were decreased were linked to polycyclic aromatic hydrocarbon degradation, suggesting that S839I mice may have higher levels of these genotoxic compounds in their gut, which could predispose them to carcinogenesis.

In contrast, pathways linked to chemical carcinogenesis were reduced (Fig. 5 F). In summary, significant differences in the microbiome of S839I mice resemble changes seen in IBD and FGDS patients (Tronstad et al., 2017) and more recent data related to the fecal microbiota seen in microscopic colitis patients (Hertz et al., 2021). Therefore, the underlying disturbances in colonic epithelial function and/or an imbalance in fluid and ion secretion due to the activating Gucy2c mutation has profound effects on the gut flora. We compared global gene expression changes in the colon of wild type and S839I mice by RNA-seq analysis, hypothesizing that the pattern of gene expression may explain the susceptibility to inflammation seen in patients.

We identified several differentially regulated genes, with a majority being down-regulated (Fig. 6 A). Differentially expressed transcripts with adjusted false discovery rate (FDR) <. 0.05 and a log2 fold change (FC) of less than −2 and >1.5 yielded 645 down-regulated and 101 up-regulated genes (Fig. 6 A).

Ingenuity Pathway Analysis (IPA) identified canonical pathways that are perturbed in S839I mice. Strikingly, increased levels of a large number of genes regulated by IFN signaling (Barrat et al., 2019), called IFN-stimulated genes (ISGs), were observed and included Ifit1, Ifit3, Ifitm3, Tap1, Irf7, Isg15, Ido1, and Socs1 (Fig. 6 B). We validated the expression levels of these genes by RTqPCRand observed that the increase in transcript levels experimentally observed was in concert with that seen in the RNA-seq analysis (Fig. 6 C).

We then looked for evidence of altered regulation of type I, type II, and type III IFN genes that would drive expression of ISGs. No members of the IFN1 and IFNIII families were detected in the RNA-seq, and transcripts were also not identified by RTPCR (data not shown). Ifng, however, could be detected by RTPCR (Fig. 6 C), and levels were higher in S839I mice. Many of the ISGs are STAT1 targets, including Socs1, which is a negative regulator of cytokine signaling.

Stat1 was up-regulated in S839I mice as was Socs1, further validating the results seen in RNA-seq analysis (Fig. 6 C). We then looked for expression of Stat1 and its phosphorylated forms by Western blotting using extracts prepared from whole colonic tissue. A significant increase in total levels of Stat1, along with phosphorylated Stat1 (at Ser727 and Tyr701), was observed in S839I mice. An increase in total Stat1 was also seen, possibly since Stat1 autoregulates its own transcription (Fig.

6 D). The significant increase in total STAT1 could also perhaps be a consequence of direct transcriptional induction in response to elevated cGMP levels. Further, levels of Isg15 (Fig. 6 E), Tap1 (Fig. 6 F), and Ido1 proteins (Fig.

6 G), which are all regulated by Stat1 and whose transcripts were increased in S839I mice (Fig. 6 C), were increased. Notably, increased STAT1 activity is associated with severity of disease in IBD in patients (Cordes et al., 2020. Schreiber et al., 2002). Given the increase in total Stat1 seen in S839I mice, it is possible that a fraction may remain unphosphorylated.

Induction of ISGs mediated by unphosphorylated STAT1, as part of a tripartite transcription complex of STAT1, STAT2, and IRF9, was reported earlier in colonic cell lines (Wang et al., 2017). Indeed, RNA-seq analysis indicated that Stat2 and Irf9 were also up-regulated in S839I mice. Expression of ISGs by unphosphorylated STAT1 is proposed to be a priming mechanism to overcome microbial (Cheon et al., 2013). The enhanced expression of ISGs in S839I mice may be a cause of the baseline pathology and consequent susceptibility to severe colitis we report here (Fig. 4).

We therefore used the Analysis Match function in IPA to compare altered gene expression seen in S839I mice with that of mice following DSS treatment. A significant number of genes showed the same trend of regulation in S839I mice and mice treated with DSS (Fig. 7), indicating that the inflammatory signatures following DSS treatment appeared to be already altered in S839I mice. IPA predicts a z-score of activation or inhibition of gene expression controlled by upstream regulators. Here again, common upstream regulators with comparable activation z-scores were found in S839I mice and those with colitis induced by DSS (Fig.

7). Finally, upstream regulators in our dataset showed similar predicted z-scores to those seen in colonic biopsies from colitis patients (Zhao et al., 2015. Fig. 7), validating that these S839I mice can indeed serve as a preclinical model to study gut inflammation in humans mediated by hyperactive GUCY2C mutations (Crowley et al., 2020). Here, we describe a novel preclinical model to understand the underlying biological mechanisms seen in patients with rare mutations in GUCY2C.

Our results show that hyperactivation of GC-C results in loss of overall homeostasis, fluid-ion imbalance, gut microbiota dysbiosis, and susceptibility to colitis. Since there is growing evidence that interorgan communication is the basis for the overall phenotype observed in organisms and FGDS patients harbored the activating mutation in all tissues where GC-C is expressed, we chose to develop a whole-body knockin model. The increased level of cGMP in IECs in S839I mice (Fig. 2 D) results in an increase in small intestinal transit via activation of Cftr (Fig. 3, C and D).

Inhibition of Nhe3 by tenapanor also resulted in enhanced transit (Fig. 3 I). A significant decrease in transcript as well as protein levels of Nhe3 was seen in S839I mice (Fig. 3, B and E). Since lower Nhe3 expression would reduce Na+ uptake by the epithelial cell, an increase in luminal sodium in the colon and the feces was seen, but not in the ileum (Fig.

3, G and H). Nhe3−/− mice displayed diarrhea with impaired fluid absorption, higher luminal sodium ion content in the intestine, and alkalization of the intestinal lumen (Schultheis et al., 1998. Xue et al., 2020), as we see here (Fig. 3 F). While Nhe3 has been reported to regulate sodium levels in mouse ileal tissue (Murtazina et al., 2011), more recent observations indicate that Nhe3 plays a modest role in sodium fluxes in the distal ileum (Stephens et al., 2021).

Therefore, there could be additional mechanisms by which sodium levels are maintained in the ileum. Inactivating mutations in NHE3 result in congenital sodium diarrhea due to malabsorption of sodium (Janecke et al., 2015). Altered and defective Na+ absorption is considered one of the most important factors for diarrhea in patients with IBD (Seidler et al., 2006). A decrease in expression or activity of NHE3 has been documented in mucosal biopsies from patients with IBD (Siddique et al., 2009. Yeruva et al., 2010).

Similarly, Il10−/− mice that develop spontaneous colitis show reduced Nhe3 activity in the enterocyte (Larmonier et al., 2011. Sellon et al., 1998). Therefore, reduced NHE3 activity in the gut of patients with hyperactive GUCY2C mutations could contribute to the incidence of Crohn’s-like symptoms and colitis. We recently speculated that impaired intestinal sodium transport and its effects on the microbiome could serve as a major upstream mediator of downstream pathophysiology (Prasad and Visweswariah, 2021). The reduced transcript levels and protein expression of Nhe3 in S839I mice suggests a role for cGMP (and perhaps PKGII) in reducing Nhe3 transcription.

Nhe3 transcription is positively regulated by Sp1/Egr-1 transcription factors (Malakooti et al., 2006). Parathyroid hormone–induced inhibition of Nhe3 transcription in opossum kidney proximal tubule cells was mediated by enhanced Egr-1 binding to the core Nhe3 promoter and activation of JAK-STAT3 activity (Neri et al., 2015). A recent study indicated that p38 activation in human colonic Caco2 cells was correlated with a reduction in Nhe3 transcription (Enns et al., 2020). We showed earlier that PKGII activates p38, which in turn results in the increased recruitment of Sp1 to the p21 promoter, resulting in enhanced p21 transcription (Basu et al., 2014). What remains to be explored in view of our findings described here is whether inhibition of Nhe3 transcription is a result of crosstalk between cGMP, PKGII, p38, Sp1/Egr-1, and STAT1 in IECs.

Activation of CFTR resulting in increased chloride efflux is the major cause of diarrhea during enteric s, causing increased chloride and bicarbonate secretion into the lumen of the intestine. Mice with hyperactivation of Cftr display signs of diarrhea and alkalization of intestinal lumen due to increased bicarbonate secretion (Gelfond et al., 2017). We show here that S839I mice also display alkalization of the small intestinal lumen (Fig. 3 F). The increased transit in the small intestine of S839I mice (Fig.

3 C) because of activation of Cftr and inhibition of Nhe3 suggests that CFTR may be targetable in FGDS patients. Inactivating mutations in CFTR lead to meconium ileus in the newborn (Sathe and Houwen, 2017) and intestinal obstructions in adults due to a reduction in gut motility and hydration. This mimics what is seen in patients with inactivating mutations in GUCY2C (Bose et al., 2020. Romi et al., 2012. Smith et al., 2015.

Woods et al., 2019), indicating the critical role Cftr plays downstream of GC-C signaling. FGDS patients displayed prolonged gut transit time and small intestinal dysmotility (von Volkmann et al., 2017). There are conflicting results for intestinal transit rate in patients with IBS-C (IBS with constipation) and IBS-D (IBS with diarrhea) in the literature (Ringel-Kulka et al., 2015. Roland et al., 2015), suggesting a complex regulation of gut motility by the enteric nervous system. S839I mice displayed higher frequency of bowel movements and fecal water content, suggesting diarrhea-like symptoms (Fig.

2, F and G). However, severe watery diarrhea marked by liquid stool in cage bedding and wet and discolored anogenital areas were not observed in S839I mice. This may be partly due to the genetic background of the mice. For example, C57BL/6N mice develop only modest diarrhea upon with the murine pathogen Citrobacter rodentium (Bhinder et al., 2013), whereas FVB/N mice showed severe diarrhea and mortality (Borenshtein et al., 2008). Commensal microbiome load and diversity are highly influenced by epithelial ion transport in the intestine (De Lisle, 2007.

Gurney et al., 2017. Keely et al., 2012). Loss of GC-C in mice results in gut microbiota dysbiosis with a lower abundance of Lactobacillus (Majumdar et al., 2018). Commensal Lactobacillus is known to play protective roles against inflammatory diseases by influencing T regulatory cell (T reg cell) functioning. An increased abundance of Proteobacteria with concomitant decrease in Lactobacillus is found in patients with IBD (Sartor, 2008).

FGDS patients displayed increased abundance of Enterobacteriaceae (γ Proteobacteria), which is associated with intestinal inflammation and symptoms similar to CD (Tronstad et al., 2017). Under physiological conditions, colonic epithelia undergo β-oxidation and deplete luminal oxygen, resulting in an anaerobic environment. However, during inflammation, the colonic epithelial cells lose their capacity of β-oxidation, resulting in increased luminal oxygen, microbiome dysbiosis, and Proteobacteria bloom (Hughes et al., 2017). This increase in Proteobacteria and decrease in Lactobacillus are also observed in Nhe3−/− knockout mice (Larmonier et al., 2013). Thus, the microbial dysbiosis seen in S839I mice resembles that of patients with IBD and mouse models of colitis (Fig.

5). The beneficial role of GC-C signaling during IBD is implicated from the observation that human patients with IBD display a significant decrease in transcript levels of GUCA2A, GUCA2B, and GUCY2C, and loss of these proteins is linked with the severity of the disease in patients (Brenna et al., 2015. Lan et al., 2016). We saw a similar change in our mice following DSS treatment (Fig. 4 C).

However, paradoxically, patients with the p.S840I mutation in GC-C display signs of CD, IBS, and obstruction in the ileum due to inflammation (Fiskerstrand et al., 2012). Perhaps the decreases in GC-C and ligand expression following DSS treatment are compensatory mechanisms that could counteract increased cGMP levels seen in S839I mice. In addition, pathways not directly regulated by cGMP could also be misregulated in these mice, which, in turn, could feed back into the regulation of GC-C and its ligands. Since mice lacking Nhe3 display a susceptibility to DSS-induced colitis (Kiela et al., 2009), electrolyte flux and imbalance in the intestine, coupled with alterations in the microbiome, may be the distal drivers of increased susceptibility to chemical-induced colitis (Prasad and Visweswariah, 2021). RNA-seq revealed that several genes linked to gut inflammation and colitis are misregulated in S839I mice.

Indeed, the pattern of gene expression is like that seen in the colon of mice treated with DSS (Fig. 7) and, importantly, IBD patients (Lamas et al., 2016. Zhao et al., 2015). Almost all the genes shown to be up-regulated in biopsies from active UC patients (Zhao et al., 2015) are similarly up-regulated in S839I mice. These include STAT1, IRF1, IRF9, IFIT2, IFIT3, IFITM2, OAS2, and ISG15.

We show here that a significant increase in total and phosphorylated Stat1 levels are seen in S839I mice (Fig. 6, C and D), which would contribute to the increased expression of ISGs. Due to the significant increase in total Stat1 in S839I mice, there may be a significant fraction of unphosphorylated Stat1 in the tissue that could also contribute to ISG expression. This noncanonical STAT signaling via unphosphorylated STATs or by Ser727 monophosphorylated STATs was described earlier on viral (Cheon et al., 2013). For example, phosphorylated STATs are dephosphorylated after entering the nucleus, but expression of target genes continues (Bandyopadhyay et al., 2008.

Cheon and Stark, 2009). Therefore, constitutive expression of hyperactive S839I in the gut of these mice results in a chronic state of STAT1 activation. We therefore suggest that GC-C via cGMP modulates STAT1 content and activity in the intestinal epithelium, leading to a basal inflammatory signal observed in S839I mice that is exacerbated in the presence of a colitis-inducing agent. In agreement with this is the fact that inflammation was reduced in Stat1−/− mice following DSS treatment (Bandyopadhyay et al., 2008), and an Ido1−/− mouse (Ido1 is up-regulated in S839I mice. Fig.

6, C and G) shows a reduced severity to DSS-induced colitis (Shon et al., 2015). In summary, we have developed a mouse model for FGDS and have identified GC-C as a key regulator of intestinal homeostasis and healthy gut functioning. Our results show the myriad consequences of hyperactivation of GC-C in the intestinal epithelium, including diarrhea, gut microbiota dysbiosis, and susceptibility to intestinal inflammation. This mouse model opens opportunities to study the role of cGMP in the gut and aid in the identification of various targets to inhibit hyperactive GC-C signaling using ex vivo organoid cultures. Importantly, this mutant mouse may serve as a model for ST-mediated diarrhea due to Enterotoxigenic E.

Coli , which is a cause of mortality and morbidity in children in developing countries. Gucy2cS839I/S839I (S839I) mice were generated via FLP-FRT recombination by Taconic Denmark (Fig. S1 A). Briefly, a p.S839I mutation was introduced in exon 22, and a puromycin cassette flanked with FRT sites was introduced in intron 21. Targeting vectors were generated using BAC clones from the C57BL/6J RPCIB-731 BAC library and were transfected into the Taconic Artemis C57BL/6N Tac ES (embryonic stem) cell line.

Homologous recombinant clones were selected using positive (PuroR) and negative (Thymidine kinase) selection. Following generation of clones, superovulated BALB/c females were mated with BALB/c males. Blastocysts were isolated from the uterus at 3.5 d after coitum. The blastocysts were microinjected with C57BL/6NTac ES cells with p.S839I mutation in Gucy2c. After recovery, eight injected blastocysts were transferred to each uterine horn of 2.5 d after coitum, pseudopregnant NMRI females.

Chimerism was measured by coat color contribution of ES cells to the BALB/c host (black/white). Chimeric mice were bred to C57BL/6N Tac females. C57BL/6N Tac female mating partners were mutant for the presence of a recombinase gene (Flp-Deleter). Germline transmission of the point mutation was identified by the presence of black C57BL/6N Tac offspring. Two breeding pairs of homozygous mice were received and backcrossed with C57BL/6N Tac wild type mice for 10 generations.

All procedures were performed in agreement with the Control and Supervision Rules, 1998 of the Ministry of Environment and Forest Act (Government of India) and the Animal Ethics Committee of the Indian Institute of Science (Approval CAF/Ethics/547/2017). All animals were bred and housed in the same vivarium. Mice were housed in a clean air facility in multiple cages and separated on the basis of sex and genotype. The temperature was maintained at 22 ± 2°C, humidity was maintained at 55% ± 10%, and the mice were maintained on a 12-h light/dark cycle. Mice had access to laboratory chow and water ad libitum.

Chow was procured from Aomin International and contained ∼24% protein, 6% oil, and 3% dietary fibers. Mice of both sexes were used for experiments unless specified. Genomic DNA for genotyping was isolated by the HotShot method (Truett et al., 2000). Briefly, 2 mm of a tail snip was incubated in 75 µl of lysis buffer (25 mM NaOH and 0.2 mM EDTA) at 95°C for 1 h. The tube was then cooled to room temperature, and 75 µl of neutralization buffer (40 mM Tris HCl, pH 5.5) was added.

Following centrifugation at 3,000 rcf for 5 min, an aliquot of the supernatant was taken for PCR. A PCR using 2 µl of the supernatant was performed with primer sets Gucy2c_27 (5′-TGA​ACA​GTA​CCC​AGG​AGA​TTA​GG-3′) and Gucy2c_28 (5′-AAC​AGT​TGC​AGA​ATC​CTT​GAG​G-3′) as indicated in Fig. S1 B. The Gucy2cS839I/S839I allele gave a 371-bp product, while the Gucy2cWT/WT allele gave a 302-bp product (Fig. S1).

For all experiments, we used the Experimental Design Assistant (RRID:SCR_017019. Https://eda.nc3rs.org.uk) to calculate the number of animals required for the experiments. Experimental Design Assistant considers the 3Rs (Replacement, Refinement, and Reduction) in its analysis and estimation of animal numbers needed for an experiment. Mice were sacrificed by CO2 asphyxiation, and ∼5 cm of the colon and 10 cm of the terminal ileum were harvested, flushed in ice-cold HBSS, cut longitudinally open, submerged in IEC dissociation buffer containing 10 mM Hepes, 1 mM EDTA, 71.5 mM β-mercaptoethanol, and 500 µM 3-isobutyl-1-methylxanthine (IBMX) to inhibit phosphodiesterases, and stored on ice. The tissues were incubated at 37°C 100 rpm for 45 min and vortexed for 30 s, and the tissue pieces were removed gently.

The tubes were centrifuged at 3,000 rpm for 10 min at 4°C. The pellet containing the IECs was washed twice with ice-cold PBS and finally resuspended in homogenization buffer containing 50 mM Hepes, pH 7.5, 100 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, 5 µg/ml soya bean trypsin inhibitor (SBTI), 5 µg/ml leupeptin, 5 µg/ml aprotinin, 2 mM PMSF, 10 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 20 mM sodium fluoride, and 500 µM IBMX. The cells were lysed by sonication at 60 cycles, 60% amplitude for 10 pulses (each pulse, 5 s. IKA Labortechnik). The suspension was centrifuged at 12,000 g for 60 min.

The pellet containing the membrane fraction was resuspended in a buffer containing 50 mM Hepes, pH 7.5, 20% glycerol, 5 µg/ml SBTI, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 2 mM PMSF, and 1 mM sodium orthovanadate, and protein concentration was estimated. Membrane preparations were used for 125I-labeled STY72F binding assay, as described earlier (Saha et al., 2009), and Western blot analysis. For cGMP estimation, the isolated and intact cells prepared from ∼5 cm of the colon or 10 cm of the terminal ileum were resuspended in PBS, and an aliquot of the suspension of cells was taken for protein estimation by Bradford assay (Bradford, 1976). Cells were harvested by centrifugation, resuspended in 0.1 N HCl, and heated at 95°C for 5 min. The mixture was then centrifuged at 17,000 rcf for 10 min at 4°C.

The supernatant was collected, and cGMP levels were estimated using a cGMP ELISA kit (Cayman Chemicals). Cyclic GMP levels were normalized to the amount of protein taken for the cGMP ELISA. 8-wk-old male wild type or S839I mice were sacrificed by CO2 asphyxiation, and 3 cm of the distal colon was harvested and snap-frozen in liquid nitrogen. The frozen tissue was crushed to a powder in liquid nitrogen using a mortar and pestle. The powdered tissue was transferred to homogenization buffer containing 50 mM Hepes, pH 7.5, 100 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, 5 µg/ml SBTI, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, 10 mM sodium orthovanadate, 1 mM sodium pyrophosphate, and 20 mM sodium fluoride.

The extract was then subjected to sonication at 60 cycles, 60% amplitude (each pulse, 5 s. IKA Labortechnik) followed by centrifugation at 12,000 g for 60 min. The pellet containing the membrane fraction was resuspended in a buffer containing 50 mM Hepes, pH 7.5, 20% glycerol, 5 µg/ml SBTI, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate. The supernatant containing the cytosol was collected. Protein concentrations in membrane and cytosolic fractions were estimated by the modified Bradford assay (Bradford, 1976).

Both membrane and cytosolic fractions were used for Western blot analysis, depending on the cellular localization of the antigen being tested. Membrane or cytosolic proteins from HEK293E cells, mouse IECs, or colonic tissue extracts were resolved on polyacrylamide gels (SDS-PAGE) and transferred onto Immun-Blot polyvinylidene fluoride membrane (Bio-Rad) in transfer buffer (25 mM Tris base, 192 mM glycine, and 20% methanol, pH 8.3) at −160 V and 200 mA for 120 min. The polyvinylidene fluoride membranes were rinsed in 10 mM Tris-Cl, pH 7.2, 100 mM NaCl, and 0.1% Tween 20 (TBS-T) and blocked for 1 h at room temperature using 5% blocking solution (GE Healthcare) prepared in TBS-T. The membrane was incubated with indicated antibodies overnight (12–14 h) at 4°C followed by three washes with TBS-T. The membrane was then incubated with anti-mouse IgG (at 1:6,000 dilution) or anti-rabbit IgG (at 1:30,000 dilution) conjugated to horseradish peroxidase for 1 h at room temperature, followed by three washes with TBS-T.

Immunoreactive bands were visualized by chemiluminescence detected using Immobilon reagent (Millipore) on a Chemidoc XRS+ (Bio-Rad) imaging system. Anti-villin and anti-Na+/K+ adenosine triphosphatase antibodies were used at a dilution of 1:5,000. Anti-pSTAT1 Ser 727, anti-pSTAT1 Tyr701, anti-STAT1, anti-TAP1, anti-ISG15, and anti–β-actin antibodies were used at a dilution of 1:1,000. Anti-Ido1 antibody was used at a dilution of 1:4,000. Anti-NHE3 antibody was used at a dilution of 1:2,000.

Anti-PKGII antibody was used at a dilution of 1:2,000. The sources of all commercial antibodies are shown in Table S2. GCC:4B11 monoclonal antibody was raised against the kinase homology domain of human GC-C and is available in the laboratory. The supernatant from cultured cells was used for Western Blot analysis at a dilution of 1:100. The frequency of bowel movements was determined as previously described (De Palma et al., 2015).

4-wk-old mice were used, and the experiment was performed between 8:00 a.m. And 9:00 a.m. Mice were placed in fresh autoclaved cages without any bedding material, and the number of pellets passed in the first 10 min was recorded as the frequency of bowel movements. Experiments were performed three times over the course of a year on independent litters. To determine total gut transit time, nonfasted mice (7–8 wk old) were gavaged with 200 µl of nonabsorbable marker dye (6% wt/vol of carmine red in filter-sterilized 0.5% methylcellulose).

The mice were housed individually in clean cages without bedding material with access to food and water. The mice were checked for output at 15-min intervals. The time taken for the first fecal pellet with carmine red to be passed was recorded as the total gut transit time. Experiments were performed three times over the course of a year on independent litters. Small intestinal transit rate and GE were estimated using the GC and GE parameters as previously described (Sobczak et al., 2014) with a few modifications.

Briefly, 8–12-wk-old mice were fasted overnight with free access to water. On the day of the experiment, mice were weighed and orally gavaged with 200 µl of a filter-sterilized marker dye mixture (50 mg/100 ml phenol red in 0.5% methylcellulose). 30 min after marker dye administration, mice were sacrificed, the gastrointestinal tract was isolated and kept chilled to reduce further peristalsis, and dissection was conducted as fast as possible. For the GC analysis, the small intestine was measured and divided into 10 equal parts. The intestinal segments were transferred to a tube containing 1 ml of distilled water and homogenized such that the intestinal contents were released into the water.

The tubes were vortexed gently and centrifuged at 3,000 g for 5 min. Following this, 250 µl of the supernatant was transferred to another fresh tube containing 250 µl of 1N NaOH, and color was allowed to develop. The intensity of color was calorimetrically measured at 560 nm using a spectrophotometer (Tecan Infinite M200 Pro. Tecan Switzerland). GC of the small intestine was calculated using the following formula:GC = Σ [(% A per segment×segment number)/100],with GC ranges from 1 (minimum motility) to 10 (maximum motility).

To determine the GE of mice, the stomach was dissected carefully, its contents were transferred to a tube containing 2 ml distilled water, and the mixture was vortexed gently followed by centrifugation at 3,000 g for 5 min. 500 µl of supernatant was transferred to another tube containing 500 µl of 1N NaOH to develop a maximum intensity of color. The intensity of color (200 µl) was measured at 560 nm using a spectrophotometer. The percentage of dye that was present in the small intestine out of the total dye present in the stomach and the small intestine was a reflection of GE. To test linaclotide-mediated enhancement in the small bowel transit rate, GC and GE analyses were performed using the protocol described earlier (Bryant et al., 2010) with a few modifications.

The mice were fasted overnight with unlimited access to water. Mice were weighed and administered 100 µg/kg of body weight linaclotide (Cayman Chemicals) prepared in 25 mM Tris-HCl, pH 7.5, orally. Mice were replaced in their cages for 10 min and then sacrificed, and GC and GE analyses were performed. To determine the effect of Cftr(inh)-172 (Sigma-Aldrich) on small bowel transit rate, overnight fasted mice were orally gavaged with 200 µg of Cftr(inh)-172 prepared in 10% wt/vol D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS). Control mice received the vehicle alone (Thiagarajah et al., 2004).

Mice were returned to their cages for 3 h, after which GC and GE analyses were performed. To determine the effect of the tenapanor (NHE3 inhibitor. MedChem Express) on small bowel motility, mice were fasted overnight and orally gavaged with 1 mg/kg tenapanor (McHugh et al., 2018) prepared in 10% TPGS. Control mice received the vehicle alone. Mice were returned to their cages for 2 h, after which the GC and GE analyses were performed.

Extraction of DNA from the fecal pellets of wild type (n = 11. 8 female and 3 male) and S839I (n = 10. 7 female and 3 male) mice was performed using the Fast DNA SPIN kit for soil (MP Biomedicals) according to the manufacturer’s protocol, with four bead-beating periods of 40 s. DNA concentration was normalized to 10 ng/µl by dilution with DNA elution solution (MP Biomedicals) to produce a final volume of 20 µl. DNA samples were sent to Clevergene Biocorp Pvt Ltd for PCR amplification of V3-V4 regions of 16S rRNA genes and paired-end sequencing (2 × 300 bp) on the Illumina MiSeq platform.

The V3-V4 hypervariable regions of the 16S rRNA genes were amplified using the 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) primers (Klindworth et al., 2013). The raw paired-end reads were obtained in fastq format from the Illumina MiSeq platform. Taxonomic abundance tables were generated from fastq files using the dada2 pipeline (v1.14.0) for paired-end reads (Callahan et al., 2016) in R (v3.6.1). Briefly, quality score plots of sequence reads were inspected to determine the drop-off points in quality of reads based on which of the forward and reverse reads were truncated at 270 and 230 positions, respectively. Primer sequences were removed by trimming the 22 bp from the left end of the raw reads.

Chimeric sequences were removed by using the remove Chimera Denovo function with the pooled sample inference method. Taxonomy was assigned to the chimera-free amplicon sequence variants (ASVs) using Silva database (v138, downloaded from McLaren, 2020). Data were filtered to remove ASVs assigned as mitochondria, chloroplasts, or other nonbacterial kingdoms and ASVs with less than two frequencies in total (singletons) using the phyloseq R package (McMurdie and Holmes, 2013. V1.30.0). Beta diversity measures were visualized using the principal coordinate analysis plot based on Bray-Curtis dissimilarity using the microbiome R package (Lahti et al., 2017.

V1.8.0). Relative abundance at taxonomic levels of phyla and genera were based on ASV counts normalized as percentages (100 * [×/sum(×)]). Significance of differences between Shannon diversity index was determined using Wilcoxon rank sum test (P <. 0.05) in the microbiome R package, while significant difference in relative abundance of taxa between wild type and S839I mice was tested using the Kruskal–Wallis test in STAMP statistical software (Parks et al., 2014). Prediction of the functional content of gut microbiome from 16S rRNA gene ASV count dataset and representative sequence of each ASV was performed using the Piphillin tool (Narayan et al., 2020).

In brief, this tool predicts functional attributes of microbial assemblages via direct nearest-neighbor matching between 16S rRNA gene amplicons and genomes from reference databases. Prediction was executed at 97% ID cutoff using KEGG (May 2020 release) and BioCyc22.5 reference databases. The output from Piphillin was analyzed by STAMP statistical software, using nonparametric Kruskal–Wallis test with Tukey-Kramer post hoc test (Parks et al., 2014). This paper is dedicated to the memory of Dr. Torunn Fiskerstrand, whose enthusiasm for development of this mouse model was motivating.

We thank Dr. Halvor Sommerfelt for his interest and support for this work and Dr. Avinash R. Shenoy for careful reading of the paper and critical suggestions. We acknowledge the help of Dr.

Harini Ramani and Yashika Bopanna in the mouse experiments. Support from the Department of Biotechnology, Ministry of Science and Technology, India is acknowledged (BT/PR15216/COE/34/02/2017), as well as from DBT-IISc Partnership Program Phase-II (BT/PR27952/INF/22/212/2018/21.01.2019). S.S. Visweswariah is a JC Bose National Fellow (SB/S2/JCB-18/2013) and a Margdarshi Fellow supported by the Wellcome Trust DBT India Alliance (IA/M/16/502606). Financial support from Helse Vest Norway, the Center for International Health, Department of Global Health and Primary Care, University of Bergen, Norway, and the Enteric treatment Initiative of PATH (https://www.path.org) is acknowledged toward the generation of the mouse model.

S.S. Visweswariah also acknowledges support from the Royal Society, UK, for a Collaborative Grant for Research Professors (IC60080), and funding from the Bill and Melinda Gates Grand Challenges Exploration Grant with Grant ID OPP1106646. Author contributions. V. Mishra, A.

Bose, S. Kiran, S. Banerjee, I.A. Shah, P. Chaukimath, M.M.

Reshi, and S. Srinivas performed experiments and analyzed data. A. Barman maintained and assisted in animal experimentation. V.

Mishra, A. Bose, S. Kiran, and S. Banerjee wrote initial drafts of the manuscript. And S.S.

Visweswariah conceived of the study, designed the analyses, and finalized the manuscript.Citation Claire Pujol, Anne Legrand, Livia Parodi, Priscilla Thomas, Fanny Mochel, Dario Saracino, Giulia Coarelli, Marijana Croon, Milica Popovic, Manon Valet, Nicolas Villain, Shahira Elshafie, Mahmoud Issa, Stephane Zuily, Mathilde Renaud, Cécilia Marelli-Tosi, Marine Legendre, Aurélien Trimouille, Isabelle Kemlin, Sophie Mathieu, Joseph G. Gleeson, Foudil Lamari, Daniele Galatolo, Rana Alkouri, Chantal Tse, Diana Rodriguez, Claire Ewenczyk, Florence Fellmann, Thierry Kuntzer, Emilie Blond, Khalid H. El Hachimi, Frédéric Darios, Alexandre Seyer, Anastasia D. Gazi, Patrick Giavalisco, Silvina Perin, Jean-Luc Boucher, Laurent Le Corre, Filippo M. Santorelli, Cyril Goizet, Maha S.

Zaki, Serge Picaud, Arnaud Mourier, Sophie Marie Steculorum, Cyril Mignot, Alexandra Durr, Aleksandra Trifunovic, Giovanni Stevanin. Implication of folate deficiency in CYP2U1 loss of function. J Exp Med 1 November 2021. 218 (11). E20210846.

Doi. Https://doi.org/10.1084/jem.20210846 Download citation file:.

Activating mutations in receptor guanylyl cyclase https://www.kampradmedia.de/kamagra-oral-jelly-100mg-factory-discount-prices/ C (GC-C), the target of gastrointestinal peptide hormones guanylin and uroguanylin, and bacterial heat-stable enterotoxins generic lasix online cause early-onset diarrhea and chronic inflammatory bowel disease (IBD). GC-C regulates ion and fluid secretion in the gut via cGMP production and activation of cGMP-dependent protein kinase II. We characterize a novel generic lasix online mouse model harboring an activating mutation in Gucy2c equivalent to that seen in an affected Norwegian family.

Mutant mice demonstrated elevated intestinal cGMP levels and enhanced fecal water and sodium content. Basal and linaclotide-mediated small intestinal transit was higher in mutant generic lasix online mice, and they were more susceptible to DSS-induced colitis. Fecal microbiome and gene expression analyses of colonic tissue revealed dysbiosis, up-regulation of IFN-stimulated genes, and misregulation of genes associated with human IBD and animal models of colitis.

This novel mouse model thus provides molecular insights into the multiple roles of intestinal epithelial cell cGMP, which culminate in dysbiosis and the induction of inflammation in the gut. Monogenic intestinal epithelium defects contributing to pediatric inflammatory bowel disease (IBD) have been described and are not readily amenable to current treatment regimens (Leung and Muise, generic lasix online 2018. Nambu and Muise, 2021).

Among the genes associated with very early–onset IBD are mutations in the receptor guanylyl cyclase C generic lasix online gene (GUCY2C. Bose et al., 2020. Crowley et al., 2020).

The receptor encoded by this gene, guanylyl cyclase C (GC-C), is the target generic lasix online of the gastrointestinal hormones guanylin (encoded by GUCA2A) and uroguanylin (encoded by GUCA2B. Arshad and Visweswariah, 2012. Basu et al., generic lasix online 2010).

GC-C is predominantly expressed along the gastrointestinal tract, where it regulates fluid and ion transport across the intestinal epithelium (Waldman and Camilleri, 2018). Ligand binding to GC-C results in elevated 3′5′-cyclic guanosine monophosphate (cGMP) levels in the intestinal cell and activation of cGMP-dependent protein kinase II (PKGII. Lohmann et generic lasix online al., 1997).

PKGII phosphorylates the cystic fibrosis transmembrane conductance regulator (CFTR) and the sodium-hydrogen exchanger, NHE3 (Chen et al., 2015. Golin-Bisello et al., 2005) generic lasix online. Phosphorylation of CFTR increases secretion of chloride and bicarbonate ions, while phosphorylation of NHE3 inhibits sodium uptake by the intestinal epithelial cell (IEC.

Chen et al., 2015). The ensuing osmotic imbalance across the IEC causes efflux of water from the cell required for mucus hydration and generic lasix online passage of the bolus of food along the gut (Arshad and Visweswariah, 2013). Familial GUCY2C diarrhea syndrome (FGDS) was first described in a Norwegian family where >30 individuals reported diarrhea of varying severity from infancy onward (Fiskerstrand et al., 2012).

The autosomal dominant mutation mapped to GUCY2C resulted in a change of Ser840 to isoleucine, present generic lasix online in the guanylyl cyclase domain. The mutation resulted in hyperactivation of GC-C whereby the mutant receptor elicited greater levels of cGMP on stimulation with ligands. These elevated cGMP levels presumably overactivated downstream signaling, resulting in increased fluid and ion secretion and diarrhea (Fiskerstrand et al., 2012).

Subsequently, we characterized an additional four activating mutations in unrelated children generic lasix online in Europe, who also presented with severe and debilitating diarrhea, detectable in utero as a greatly distended abdomen in the fetus (Müller et al., 2016). The mutations (Lys507Glu, Leu775Pro, Arg792Ser, and Asn850Asp) were present in different domains of the receptor, including the kinase-homology domain, the linker region, and the catalytic domain (Bose et al., 2020. Mishra et generic lasix online al., 2018).

Patients suffering from FGDS and children with de novo mutations in GUCY2C present with Crohn’s disease (CD)–like symptoms and colitis in addition to diarrhea (Fiskerstrand et al., 2012. Müller et generic lasix online al., 2016). GC-C is the target of bacterial heat-stable enterotoxins (STs) produced by enterotoxigenic Escherichia coli, one of the major causes of watery diarrhea in developing countries (Schulz et al., 1990).

The Food and Drug Administration–approved drugs linaclotide and plecanatide, which are used to treat constipation, comprise the cysteine-rich core sequence of ST or uroguanylin, respectively, and activate GC-C to induce cGMP production (Shah et al., 2018). However, no drugs are available to alleviate diarrhea mediated by GC-C, though molecules that may inhibit proteins downstream of GC-C show promise (Bijvelds et al., 2018) generic lasix online. Knockout mice for GC-C have been studied for several years (Schulz et al., 1997).

While they show no apparent signs of constipation, reports indicate that they demonstrate neurological symptoms (Brierley, generic lasix online 2012. Mann et al., 2019), and suppression of uroguanylin-mediated GC-C signaling in mice results in obesity (Valentino et al., 2011). We have demonstrated that GC-C−/− mice are more susceptible to oral Salmonella enterica enterica serovar Typhimurium (Majumdar et al., 2018).

Inactivating mutations have been reported in generic lasix online GUCY2C, with infants presenting with meconium ileus at birth. These children were the outcome of consanguinity, with mutations present in a homozygous state (Romi et al., 2012. Smith et generic lasix online al., 2015.

Woods et al., 2019). The lack of models for diarrheal disease mediated by hyperactive GC-C to investigate the link between GC-C, cGMP, and gut inflammation prompted us to develop a mutant mouse harboring a mutation in Gucy2c, equivalent to the S840I mutation found in the Norwegian family. Here, we validate this model in terms of increased frequency of passing generic lasix online watery feces, regulation of Cftr and Nhe3 activity in vivo, and enhanced susceptibility of these mice to colitis.

Analysis of the fecal microbiome in mutant mice and global colonic gene expression provided the underlying explanation for inflammation observed in patients harboring activating mutations of GC-C. Therefore, this mutant generic lasix online mouse serves as a preclinical model for GUCY2C-mediated secretory diarrhea associated with IBD and the more prevalent infectious diarrheal illness caused by GC-C activation. Transcript levels of Gucy2c and uroguanylin (Guca2b) were similar in the ileum and colon of wild type and S839I mice, while levels of guanylin (Guca2a) were significantly lower in S839I mice (Fig.

2 A). The gut is the major site of generic lasix online synthesis of guanylin and uroguanylin. Therefore, serum levels of the propeptides are reflective of levels produced in the gut.

However, we did not see a significant decrease in propeptide hormone levels in S839I generic lasix online mice sera (Fig. S3). Western blotting revealed that GC-C expression was similar in wild type and S839I mice (Fig.

2 B), as was ST binding generic lasix online to membranes prepared from epithelial cells (Fig. 2 C). The presence of GC-C harboring hyperactivating mutations in patients’ gut was hypothesized generic lasix online to result in elevated intra-epithelial cell cGMP in response to guanylin and/or uroguanylin (Fiskerstrand et al., 2012.

Müller et al., 2016). As shown in Fig. 2 D, steady-state levels of cGMP in epithelial cells were ∼5- to 10-fold generic lasix online higher in S839I mice.

Therefore, mutant mouse GC-C indeed elicited a greater response in terms of cGMP production in response to the endogenous ligands. Patients with FGDS demonstrated a delayed gut transit time due to regurgitation of gut contents in the small intestine (von Volkmann generic lasix online et al., 2017, 2016). We estimated gut transit in wild type and S839I mice but observed no change in S839I mice (Fig.

2 E) generic lasix online. Patients with activating mutations in GUCY2C pass multiple, watery stools (Fiskerstrand et al., 2012). We monitored the number of fecal pellets passed by mice in 10 min and observed that S839I mice passed a greater number of fecal pellets (Fig.

2 F) generic lasix online. The water content in feces produced by S839I mice was higher (Fig. 2 G) generic lasix online.

These phenotypes mirror the symptoms of diarrhea seen in FGDS, suggesting that S839I mice can be used to understand processes that regulate the frequent episodes of bowel evacuation seen in FGDS patients. Elevated cGMP levels in IECs stimulate enhanced chloride and bicarbonate secretion through CFTR following phosphorylation by cGMP-dependent protein kinase G II (Fig. 3 A generic lasix online.

Golin-Bisello et al., 2005. Lin et generic lasix online al., 1992). In addition, inhibitory phosphorylation of the sodium-hydrogen exchanger NHE3 (SLC9A3) results in reduced sodium ion import into cells and consequent increase in luminal and fecal sodium (Chen et al., 2015).

Transcript levels of PrkgII were reduced in the colon of S839I mice as was the level of the protein (Fig. S3 B), suggesting that some effects of cGMP in the colon could be generic lasix online PKGII independent. While Cftr transcripts were similar in both wild type and S839I mice, levels of Nhe3 were lower in both the ileum and colon in S839I mice (Fig.

3 B) generic lasix online. We measured small intestinal transit rates in wild type and S839I mice. Gastric emptying (GE) was slightly enhanced in wild type mice following treatment with the Cftr inhibitor, but not significantly, though GE is reported to be enhanced in CFTR patients (Collins et al., 1997).

No change in GE was seen in S839I mice generic lasix online (Fig. 3 C, upper panel) either in the presence or absence of a Cftr inhibitor. However, the extent of migration of the dye in the generic lasix online small intestine, as measured by the geometric center (GC), was significantly higher in S839I mice (Fig.

3 C, lower panel) treated with vehicle alone. On administration of the Cftr inhibitor, an increase in GC in wild type mice was seen (Fig. 3 C, lower panel), which agrees with the paradoxical observation that upper small intestinal transit is increased in cystic fibrosis patients (Hedsund et al., generic lasix online 2012).

Importantly, a dramatic reduction in the GC was seen in S839I mice treated with the Cftr inhibitor (Fig. 3 C, lower panel), demonstrating that the increased basal transit rate in S839I generic lasix online mice was almost solely due to CFTR activation. We then administered linaclotide to more potently activate GC-C (Bryant et al., 2010).

GE was higher in S839I mice (Fig generic lasix online. 3 D, upper panel) when mice were gavaged with buffer alone, in contrast to what was seen in mice gavaged with the vehicle used to dissolve the Cftr inhibitor (Fig. 3 C, upper panel).

GC-C and uroguanylin expression generic lasix online has been reported in the stomach of mammals, albeit at low levels (Date et al., 1999. London et al., 1997), and the increased GE could be a consequence of hyperactive GC-C in the stomach. On linaclotide treatment, the GC was increased in both wild type and S839I mice (Fig generic lasix online.

3 D, lower panel). However, transit was significantly higher in S839I mice. Therefore, ligand-mediated generic lasix online activation of hyperactive GC-C causes more rapid migration down the small intestine.

We had observed a transcriptional down-regulation of Nhe3 in both the ileum and the colon of S839I mice in comparison with wild type mice (Fig. 3 B) generic lasix online. We monitored expression of Nhe3 and saw a significant reduction in protein expression in both the ileum and colon of S839I mice (Fig.

3 E). Increased bicarbonate efflux from the cell, due to generic lasix online elevated cGMP levels and Cftr activity, along with reduced expression of Nhe3 should increase sodium levels and luminal pH along the gut. In agreement with this, luminal pH in the ileum of S839I mice was higher than in wild type mice (Fig.

3 F) generic lasix online. However, a significant increase in luminal sodium was observed only in the colon (Fig. 3 G) and was correlated with higher fecal sodium content (Fig.

3 H) generic lasix online. This suggests that Nhe3 may not be the main contributor to sodium import in the small intestine. However, down-regulation of Nhe3 coupled with inhibition of its activity due to elevated cGMP levels in the colon manifests in enhanced excretion of fecal sodium, as seen in children with hyperactivating mutations in GC-C that show congenital generic lasix online sodium secretory diarrhea (Müller et al., 2016).

We evaluated the extent to which Nhe3 is inhibited by elevated cGMP levels in the gut in S839I mice by administering tenapanor, a specific Nhe3 inhibitor. While GE was again higher in S839I mice administered vehicle alone (Fig. 3 I, upper panel), no further increase was seen in both wild type or S839I generic lasix online mice following administration of tenapanor.

However, in agreement with earlier studies (McHugh et al., 2018), administration of the inhibitor to wild type mice increased the GC (Fig. 3 I, lower generic lasix online panel). In S839I mice, the already elevated basal transit was further enhanced by tenapanor treatment (Fig.

3 I). We attribute this enhanced increase generic lasix online in small intestinal transit to the prevalent lower levels of Nhe3 present in S839I mice and efficient inhibition by tenapanor. Patients harboring activating mutations in GUCY2C present with varying degrees of inflammation in the gut (such as esophagitis, irritable bowel syndrome [IBS], CD, and ulcerative colitis [UC].

Fiskerstrand et al., 2012 generic lasix online. Müller et al., 2016). Administration of DSS to mice causes death of epithelial cells and compromises barrier function generic lasix online (Wirtz et al., 2017).

We administered DSS to mice and monitored weight loss and damage to the colon. S839I mice were more susceptible to DSS as evidenced by greater weight loss (Fig. 4 A, left panel) and higher generic lasix online disease activity index (Fig.

4 A, right panel). Colonic shortening was observed generic lasix online in both wild type and S839I mice after DSS treatment (Fig. 4 B).

Transcript levels of GC-C and guanylin are reduced in biopsies taken from human UC and CD patients (Brenna et al., 2015. Lan et al., 2016) generic lasix online. Following the induction of colitis by DSS, transcript levels of Gucy2c and Guca2a were reduced in both wild type and S839I mice (Fig.

4 C) generic lasix online. Histological evaluation of the colon in wild type and S839I mice revealed no difference in colon architecture or changes in crypt depth, indicating that IEC turnover was similar in S839I mice (Fig. S3 C).

After DSS treatment, a greater degree of crypt abscesses and destruction of generic lasix online colonic mucosa was observed in S839I mice (Fig. 4 D). Concomitant with greater mucosal damage, generic lasix online fecal lipocalin, a sensitive marker for inflammation in the gut (Chassaing et al., 2012), was increased in S839I mice (Fig.

4 E). Taken together, our results show that S839I mice harboring an activating mutation in Gucy2c reveal roles of cGMP in regulating gut function and enhanced colonic susceptibility to damage in a colitis model. Notably, GC-C knockout generic lasix online mice are resistant to DSS-induced colitis (Steinbrecher et al., 2011).

To explore global changes seen in the gut because of the presence of hyperactive GC-C, we took unbiased approaches by characterizing the fecal microbiome and the transcriptome in colonic tissue. The altered pH and generic lasix online sodium ion concentrations in the gut lumen may result in dysbiosis that could predispose to colitis. We therefore performed 16S ribosomal RNA (rRNA) gene amplicon sequencing of fecal samples collected from wild type and S839I mice.

Unconstrained ordination through principal component analysis with Bray-Curtis dissimilarity metrics displayed a clear separation between the two sets of mice (analysis of similarities P value = 0.001. Fig. 5 A).

There was a reproducible trend of reduced α diversity (P <. 0.1) in the fecal microbiome of S839I mice (Fig. 5 B), and relative abundance plots demonstrated differences at the phylum- and genus-levels (Fig.

5 C). An increased abundance of potential opportunistic pathogens (Anaeroplasma, Desulfovibrio, Mucispirillum, and Paraprevotella) was seen in S839I (Fig. 5 D).

Members of the genus Paraprevotella are associated with colonic CD and produce succinic acid, increased levels of which are reported to be associated with microbiome dysbiosis and intestinal inflammation in patients with IBD and animal models of chronic colitis (Macias-Ceja et al., 2019. Walters et al., 2014). The genus Mucispirillum is also significantly higher in S839I mice (Fig.

5 D). Exposure of Nod2−/−Cybb−/− C57BL/6 mice to a mucus-dwelling Gram-negative pathobiont of rodents, Mucispirillum schaedleri, has been reported to trigger the development of CD-like colitis (Caruso et al., 2019). Desulfovibrio was significantly enriched in S839I mice as seen in the colonic mucosal and fecal microbiome of UC and CD patients (Rowan et al., 2010).

Levels of protective bacteria such as Colidextribacter, Dorea (short-chain fatty acid producers), Dubosiella, and Lactobacillus (possessing anti-inflammatory properties) were significantly reduced in S839I mice (Fig. 5 E). Colidextribacter and Dorea belong to Clostridiales cluster IV and Clostridium cluster XIVa in the phylum Firmicutes, respectively.

These taxa are known to produce short-chain fatty acid and are reported to be less abundant in the ileal biopsy and fecal samples isolated from CD patients (Nagao-Kitamoto and Kamada, 2017). Lactobacillus strains restore the commensals and gut homeostasis in intestinal disorders (Blaser, 2014). Therefore, the lower abundance of these genera in S839I mice suggests that these animals may be more susceptible to environment-induced colitis and inflammation in the gut, as reported in FGDS patients (Fiskerstrand et al., 2012).

Analysis of predicted functional consequences of the variation in abundance of taxa between the mice (Narayan et al., 2020) indicated significant differences in 28 KEGG pathways (Table S3). Those linked to host immunity were enriched in S839I mice and included NOD-like receptor signaling, antigen processing and presentation, IL17 signaling, and Th17 cell differentiation (Fig. 5 F).

KEGG pathways for bacterial chemotaxis and flagellar assembly, both associated with cell motility in the microbiome, were significantly higher in S839I mice (Table S3). Pathways that were decreased were linked to polycyclic aromatic hydrocarbon degradation, suggesting that S839I mice may have higher levels of these genotoxic compounds in their gut, which could predispose them to carcinogenesis. In contrast, pathways linked to chemical carcinogenesis were reduced (Fig.

5 F). In summary, significant differences in the microbiome of S839I mice resemble changes seen in IBD and FGDS patients (Tronstad et al., 2017) and more recent data related to the fecal microbiota seen in microscopic colitis patients (Hertz et al., 2021). Therefore, the underlying disturbances in colonic epithelial function and/or an imbalance in fluid and ion secretion due to the activating Gucy2c mutation has profound effects on the gut flora.

We compared global gene expression changes in the colon of wild type and S839I mice by RNA-seq analysis, hypothesizing that the pattern of gene expression may explain the susceptibility to inflammation seen in patients. We identified several differentially regulated genes, with a majority being down-regulated (Fig. 6 A).

Differentially expressed transcripts with adjusted false discovery rate (FDR) <. 0.05 and a log2 fold change (FC) of less than −2 and >1.5 yielded 645 down-regulated and 101 up-regulated genes (Fig. 6 A).

Ingenuity Pathway Analysis (IPA) identified canonical pathways that are perturbed in S839I mice. Strikingly, increased levels of a large number of genes regulated by IFN signaling (Barrat et al., 2019), called IFN-stimulated genes (ISGs), were observed and included Ifit1, Ifit3, Ifitm3, Tap1, Irf7, Isg15, Ido1, and Socs1 (Fig. 6 B).

We validated the expression levels of these genes by RTqPCRand observed that the increase in transcript levels experimentally observed was in concert with that seen in the RNA-seq analysis (Fig. 6 C). We then looked for evidence of altered regulation of type I, type II, and type III IFN genes that would drive expression of ISGs.

No members of the IFN1 and IFNIII families were detected in the RNA-seq, and transcripts were also not identified by RTPCR (data not shown). Ifng, however, could be detected by RTPCR (Fig. 6 C), and levels were higher in S839I mice.

Many of the ISGs are STAT1 targets, including Socs1, which is a negative regulator of cytokine signaling. Stat1 was up-regulated in S839I mice as was Socs1, further validating the results seen in RNA-seq analysis (Fig. 6 C).

We then looked for expression of Stat1 and its phosphorylated forms by Western blotting using extracts prepared from whole colonic tissue. A significant increase in total levels of Stat1, along with phosphorylated Stat1 (at Ser727 and Tyr701), was observed in S839I mice. An increase in total Stat1 was also seen, possibly since Stat1 autoregulates its own transcription (Fig.

6 D). The significant increase in total STAT1 could also perhaps be a consequence of direct transcriptional induction in response to elevated cGMP levels. Further, levels of Isg15 (Fig.

6 E), Tap1 (Fig. 6 F), and Ido1 proteins (Fig. 6 G), which are all regulated by Stat1 and whose transcripts were increased in S839I mice (Fig.

6 C), were increased. Notably, increased STAT1 activity is associated with severity of disease in IBD in patients (Cordes et al., 2020. Schreiber et al., 2002).

Given the increase in total Stat1 seen in S839I mice, it is possible that a fraction may remain unphosphorylated. Induction of ISGs mediated by unphosphorylated STAT1, as part of a tripartite transcription complex of STAT1, STAT2, and IRF9, was reported earlier in colonic cell lines (Wang et al., 2017). Indeed, RNA-seq analysis indicated that Stat2 and Irf9 were also up-regulated in S839I mice.

Expression of ISGs by unphosphorylated STAT1 is proposed to be a priming mechanism to overcome microbial (Cheon et al., 2013). The enhanced expression of ISGs in S839I mice may be a cause of the baseline pathology and consequent susceptibility to severe colitis we report here (Fig. 4).

We therefore used the Analysis Match function in IPA to compare altered gene expression seen in S839I mice with that of mice following DSS treatment. A significant number of genes showed the same trend of regulation in S839I mice and mice treated with DSS (Fig. 7), indicating that the inflammatory signatures following DSS treatment appeared to be already altered in S839I mice.

IPA predicts a z-score of activation or inhibition of gene expression controlled by upstream regulators. Here again, common upstream regulators with comparable activation z-scores were found in S839I mice and those with colitis induced by DSS (Fig. 7).

Finally, upstream regulators in our dataset showed similar predicted z-scores to those seen in colonic biopsies from colitis patients (Zhao et al., 2015. Fig. 7), validating that these S839I mice can indeed serve as a preclinical model to study gut inflammation in humans mediated by hyperactive GUCY2C mutations (Crowley et al., 2020).

Here, we describe a novel preclinical model to understand the underlying biological mechanisms seen in patients with rare mutations in GUCY2C. Our results show that hyperactivation of GC-C results in loss of overall homeostasis, fluid-ion imbalance, gut microbiota dysbiosis, and susceptibility to colitis. Since there is growing evidence that interorgan communication is the basis for the overall phenotype observed in organisms and FGDS patients harbored the activating mutation in all tissues where GC-C is expressed, we chose to develop a whole-body knockin model.

The increased level of cGMP in IECs in S839I mice (Fig. 2 D) results in an increase in small intestinal transit via activation of Cftr (Fig. 3, C and D).

Inhibition of Nhe3 by tenapanor also resulted in enhanced transit (Fig. 3 I). A significant decrease in transcript as well as protein levels of Nhe3 was seen in S839I mice (Fig.

3, B and E). Since lower Nhe3 expression would reduce Na+ uptake by the epithelial cell, an increase in luminal sodium in the colon and the feces was seen, but not in the ileum (Fig. 3, G and H).

Nhe3−/− mice displayed diarrhea with impaired fluid absorption, higher luminal sodium ion content in the intestine, and alkalization of the intestinal lumen (Schultheis et al., 1998. Xue et al., 2020), as we see here (Fig. 3 F).

While Nhe3 has been reported to regulate sodium levels in mouse ileal tissue (Murtazina et al., 2011), more recent observations indicate that Nhe3 plays a modest role in sodium fluxes in the distal ileum (Stephens et al., 2021). Therefore, there could be additional mechanisms by which sodium levels are maintained in the ileum. Inactivating mutations in NHE3 result in congenital sodium diarrhea due to malabsorption of sodium (Janecke et al., 2015).

Altered and defective Na+ absorption is considered one of the most important factors for diarrhea in patients with IBD (Seidler et al., 2006). A decrease in expression or activity of NHE3 has been documented in mucosal biopsies from patients with IBD (Siddique et al., 2009. Yeruva et al., 2010).

Similarly, Il10−/− mice that develop spontaneous colitis show reduced Nhe3 activity in the enterocyte (Larmonier et al., 2011. Sellon et al., 1998). Therefore, reduced NHE3 activity in the gut of patients with hyperactive GUCY2C mutations could contribute to the incidence of Crohn’s-like symptoms and colitis.

We recently speculated that impaired intestinal sodium transport and its effects on the microbiome could serve as a major upstream mediator of downstream pathophysiology (Prasad and Visweswariah, 2021). The reduced transcript levels and protein expression of Nhe3 in S839I mice suggests a role for cGMP (and perhaps PKGII) in reducing Nhe3 transcription. Nhe3 transcription is positively regulated by Sp1/Egr-1 transcription factors (Malakooti et al., 2006).

Parathyroid hormone–induced inhibition of Nhe3 transcription in opossum kidney proximal tubule cells was mediated by enhanced Egr-1 binding to the core Nhe3 promoter and activation of JAK-STAT3 activity (Neri et al., 2015). A recent study indicated that p38 activation in human colonic Caco2 cells was correlated with a reduction in Nhe3 transcription (Enns et al., 2020). We showed earlier that PKGII activates p38, which in turn results in the increased recruitment of Sp1 to the p21 promoter, resulting in enhanced p21 transcription (Basu et al., 2014).

What remains to be explored in view of our findings described here is whether inhibition of Nhe3 transcription is a result of crosstalk between cGMP, PKGII, p38, Sp1/Egr-1, and STAT1 in IECs. Activation of CFTR resulting in increased chloride efflux is the major cause of diarrhea during enteric s, causing increased chloride and bicarbonate secretion into the lumen of the intestine. Mice with hyperactivation of Cftr display signs of diarrhea and alkalization of intestinal lumen due to increased bicarbonate secretion (Gelfond et al., 2017).

We show here that S839I mice also display alkalization of the small intestinal lumen (Fig. 3 F). The increased transit in the small intestine of S839I mice (Fig.

3 C) because of activation of Cftr and inhibition of Nhe3 suggests that CFTR may be targetable in FGDS patients. Inactivating mutations in CFTR lead to meconium ileus in the newborn (Sathe and Houwen, 2017) and intestinal obstructions in adults due to a reduction in gut motility and hydration. This mimics what is seen in patients with inactivating mutations in GUCY2C (Bose et al., 2020.

Romi et al., 2012. Smith et al., 2015. Woods et al., 2019), indicating the critical role Cftr plays downstream of GC-C signaling.

FGDS patients displayed prolonged gut transit time and small intestinal dysmotility (von Volkmann et al., 2017). There are conflicting results for intestinal transit rate in patients with IBS-C (IBS with constipation) and IBS-D (IBS with diarrhea) in the literature (Ringel-Kulka et al., 2015. Roland et al., 2015), suggesting a complex regulation of gut motility by the enteric nervous system.

S839I mice displayed higher frequency of bowel movements and fecal water content, suggesting diarrhea-like symptoms (Fig. 2, F and G). However, severe watery diarrhea marked by liquid stool in cage bedding and wet and discolored anogenital areas were not observed in S839I mice.

This may be partly due to the genetic background of the mice. For example, C57BL/6N mice develop only modest diarrhea upon with the murine pathogen Citrobacter rodentium (Bhinder et al., 2013), whereas FVB/N mice showed severe diarrhea and mortality (Borenshtein et al., 2008). Commensal microbiome load and diversity are highly influenced by epithelial ion transport in the intestine (De Lisle, 2007.

Gurney et al., 2017. Keely et al., 2012). Loss of GC-C in mice results in gut microbiota dysbiosis with a lower abundance of Lactobacillus (Majumdar et al., 2018).

Commensal Lactobacillus is known to play protective roles against inflammatory diseases by influencing T regulatory cell (T reg cell) functioning. An increased abundance of Proteobacteria with concomitant decrease in Lactobacillus is found in patients with IBD (Sartor, 2008). FGDS patients displayed increased abundance of Enterobacteriaceae (γ Proteobacteria), which is associated with intestinal inflammation and symptoms similar to CD (Tronstad et al., 2017).

Under physiological conditions, colonic epithelia undergo β-oxidation and deplete luminal oxygen, resulting in an anaerobic environment. However, during inflammation, the colonic epithelial cells lose their capacity of β-oxidation, resulting in increased luminal oxygen, microbiome dysbiosis, and Proteobacteria bloom (Hughes et al., 2017). This increase in Proteobacteria and decrease in Lactobacillus are also observed in Nhe3−/− knockout mice (Larmonier et al., 2013).

Thus, the microbial dysbiosis seen in S839I mice resembles that of patients with IBD and mouse models of colitis (Fig. 5). The beneficial role of GC-C signaling during IBD is implicated from the observation that human patients with IBD display a significant decrease in transcript levels of GUCA2A, GUCA2B, and GUCY2C, and loss of these proteins is linked with the severity of the disease in patients (Brenna et al., 2015.

Lan et al., 2016). We saw a similar change in our mice following DSS treatment (Fig. 4 C).

However, paradoxically, patients with the p.S840I mutation in GC-C display signs of CD, IBS, and obstruction in the ileum due to inflammation (Fiskerstrand et al., 2012). Perhaps the decreases in GC-C and ligand expression following DSS treatment are compensatory mechanisms that could counteract increased cGMP levels seen in S839I mice. In addition, pathways not directly regulated by cGMP could also be misregulated in these mice, which, in turn, could feed back into the regulation of GC-C and its ligands.

Since mice lacking Nhe3 display a susceptibility to DSS-induced colitis (Kiela et al., 2009), electrolyte flux and imbalance in the intestine, coupled with alterations in the microbiome, may be the distal drivers of increased susceptibility to chemical-induced colitis (Prasad and Visweswariah, 2021). RNA-seq revealed that several genes linked to gut inflammation and colitis are misregulated in S839I mice. Indeed, the pattern of gene expression is like that seen in the colon of mice treated with DSS (Fig.

7) and, importantly, IBD patients (Lamas et al., 2016. Zhao et al., 2015). Almost all the genes shown to be up-regulated in biopsies from active UC patients (Zhao et al., 2015) are similarly up-regulated in S839I mice.

These include STAT1, IRF1, IRF9, IFIT2, IFIT3, IFITM2, OAS2, and ISG15. We show here that a significant increase in total and phosphorylated Stat1 levels are seen in S839I mice (Fig. 6, C and D), which would contribute to the increased expression of ISGs.

Due to the significant increase in total Stat1 in S839I mice, there may be a significant fraction of unphosphorylated Stat1 in the tissue that could also contribute to ISG expression. This noncanonical STAT signaling via unphosphorylated STATs or by Ser727 monophosphorylated STATs was described earlier on viral (Cheon et al., 2013). For example, phosphorylated STATs are dephosphorylated after entering the nucleus, but expression of target genes continues (Bandyopadhyay et al., 2008.

Cheon and Stark, 2009). Therefore, constitutive expression of hyperactive S839I in the gut of these mice results in a chronic state of STAT1 activation. We therefore suggest that GC-C via cGMP modulates STAT1 content and activity in the intestinal epithelium, leading to a basal inflammatory signal observed in S839I mice that is exacerbated in the presence of a colitis-inducing agent.

In agreement with this is the fact that inflammation was reduced in Stat1−/− mice following DSS treatment (Bandyopadhyay et al., 2008), and an Ido1−/− mouse (Ido1 is up-regulated in S839I mice. Fig. 6, C and G) shows a reduced severity to DSS-induced colitis (Shon et al., 2015).

In summary, we have developed a mouse model for FGDS and have identified GC-C as a key regulator of intestinal homeostasis and healthy gut functioning. Our results show the myriad consequences of hyperactivation of GC-C in the intestinal epithelium, including diarrhea, gut microbiota dysbiosis, and susceptibility to intestinal inflammation. This mouse model opens opportunities to study the role of cGMP in the gut and aid in the identification of various targets to inhibit hyperactive GC-C signaling using ex vivo organoid cultures.

Importantly, this mutant mouse may serve as a model for ST-mediated diarrhea due to Enterotoxigenic E. Coli , which is a cause of mortality and morbidity in children in developing countries. Gucy2cS839I/S839I (S839I) mice were generated via FLP-FRT recombination by Taconic Denmark (Fig.

S1 A). Briefly, a p.S839I mutation was introduced in exon 22, and a puromycin cassette flanked with FRT sites was introduced in intron 21. Targeting vectors were generated using BAC clones from the C57BL/6J RPCIB-731 BAC library and were transfected into the Taconic Artemis C57BL/6N Tac ES (embryonic stem) cell line.

Homologous recombinant clones were selected using positive (PuroR) and negative (Thymidine kinase) selection. Following generation of clones, superovulated BALB/c females were mated with BALB/c males. Blastocysts were isolated from the uterus at 3.5 d after coitum.

The blastocysts were microinjected with C57BL/6NTac ES cells with p.S839I mutation in Gucy2c. After recovery, eight injected blastocysts were transferred to each uterine horn of 2.5 d after coitum, pseudopregnant NMRI females. Chimerism was measured by coat color contribution of ES cells to the BALB/c host (black/white).

Chimeric mice were bred to C57BL/6N Tac females. C57BL/6N Tac female mating partners were mutant for the presence of a recombinase gene (Flp-Deleter). Germline transmission of the point mutation was identified by the presence of black C57BL/6N Tac offspring.

Two breeding pairs of homozygous mice were received and backcrossed with C57BL/6N Tac wild type mice for 10 generations. All procedures were performed in agreement with the Control and Supervision Rules, 1998 of the Ministry of Environment and Forest Act (Government of India) and the Animal Ethics Committee of the Indian Institute of Science (Approval CAF/Ethics/547/2017). All animals were bred and housed in the same vivarium.

Mice were housed in a clean air facility in multiple cages and separated on the basis of sex and genotype. The temperature was maintained at 22 ± 2°C, humidity was maintained at 55% ± 10%, and the mice were maintained on a 12-h light/dark cycle. Mice had access to laboratory chow and water ad libitum.

Chow was procured from Aomin International and contained ∼24% protein, 6% oil, and 3% dietary fibers. Mice of both sexes were used for experiments unless specified. Genomic DNA for genotyping was isolated by the HotShot method (Truett et al., 2000).

Briefly, 2 mm of a tail snip was incubated in 75 µl of lysis buffer (25 mM NaOH and 0.2 mM EDTA) at 95°C for 1 h. The tube was then cooled to room temperature, and 75 µl of neutralization buffer (40 mM Tris HCl, pH 5.5) was added. Following centrifugation at 3,000 rcf for 5 min, an aliquot of the supernatant was taken for PCR.

A PCR using 2 µl of the supernatant was performed with primer sets Gucy2c_27 (5′-TGA​ACA​GTA​CCC​AGG​AGA​TTA​GG-3′) and Gucy2c_28 (5′-AAC​AGT​TGC​AGA​ATC​CTT​GAG​G-3′) as indicated in Fig. S1 B. The Gucy2cS839I/S839I allele gave a 371-bp product, while the Gucy2cWT/WT allele gave a 302-bp product (Fig.

S1). For all experiments, we used the Experimental Design Assistant (RRID:SCR_017019. Https://eda.nc3rs.org.uk) to calculate the number of animals required for the experiments.

Experimental Design Assistant considers the 3Rs (Replacement, Refinement, and Reduction) in its analysis and estimation of animal numbers needed for an experiment. Mice were sacrificed by CO2 asphyxiation, and ∼5 cm of the colon and 10 cm of the terminal ileum were harvested, flushed in ice-cold HBSS, cut longitudinally open, submerged in IEC dissociation buffer containing 10 mM Hepes, 1 mM EDTA, 71.5 mM β-mercaptoethanol, and 500 µM 3-isobutyl-1-methylxanthine (IBMX) to inhibit phosphodiesterases, and stored on ice. The tissues were incubated at 37°C 100 rpm for 45 min and vortexed for 30 s, and the tissue pieces were removed gently.

The tubes were centrifuged at 3,000 rpm for 10 min at 4°C. The pellet containing the IECs was washed twice with ice-cold PBS and finally resuspended in homogenization buffer containing 50 mM Hepes, pH 7.5, 100 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, 5 µg/ml soya bean trypsin inhibitor (SBTI), 5 µg/ml leupeptin, 5 µg/ml aprotinin, 2 mM PMSF, 10 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 20 mM sodium fluoride, and 500 µM IBMX. The cells were lysed by sonication at 60 cycles, 60% amplitude for 10 pulses (each pulse, 5 s.

IKA Labortechnik). The suspension was centrifuged at 12,000 g for 60 min. The pellet containing the membrane fraction was resuspended in a buffer containing 50 mM Hepes, pH 7.5, 20% glycerol, 5 µg/ml SBTI, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 2 mM PMSF, and 1 mM sodium orthovanadate, and protein concentration was estimated.

Membrane preparations were used for 125I-labeled STY72F binding assay, as described earlier (Saha et al., 2009), and Western blot analysis. For cGMP estimation, the isolated and intact cells prepared from ∼5 cm of the colon or 10 cm of the terminal ileum were resuspended in PBS, and an aliquot of the suspension of cells was taken for protein estimation by Bradford assay (Bradford, 1976). Cells were harvested by centrifugation, resuspended in 0.1 N HCl, and heated at 95°C for 5 min.

The mixture was then centrifuged at 17,000 rcf for 10 min at 4°C. The supernatant was collected, and cGMP levels were estimated using a cGMP ELISA kit (Cayman Chemicals). Cyclic GMP levels were normalized to the amount of protein taken for the cGMP ELISA.

8-wk-old male wild type or S839I mice were sacrificed by CO2 asphyxiation, and 3 cm of the distal colon was harvested and snap-frozen in liquid nitrogen. The frozen tissue was crushed to a powder in liquid nitrogen using a mortar and pestle. The powdered tissue was transferred to homogenization buffer containing 50 mM Hepes, pH 7.5, 100 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, 5 µg/ml SBTI, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, 10 mM sodium orthovanadate, 1 mM sodium pyrophosphate, and 20 mM sodium fluoride.

The extract was then subjected to sonication at 60 cycles, 60% amplitude (each pulse, 5 s. IKA Labortechnik) followed by centrifugation at 12,000 g for 60 min. The pellet containing the membrane fraction was resuspended in a buffer containing 50 mM Hepes, pH 7.5, 20% glycerol, 5 µg/ml SBTI, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate.

The supernatant containing the cytosol was collected. Protein concentrations in membrane and cytosolic fractions were estimated by the modified Bradford assay (Bradford, 1976). Both membrane and cytosolic fractions were used for Western blot analysis, depending on the cellular localization of the antigen being tested.

Membrane or cytosolic proteins from HEK293E cells, mouse IECs, or colonic tissue extracts were resolved on polyacrylamide gels (SDS-PAGE) and transferred onto Immun-Blot polyvinylidene fluoride membrane (Bio-Rad) in transfer buffer (25 mM Tris base, 192 mM glycine, and 20% methanol, pH 8.3) at −160 V and 200 mA for 120 min. The polyvinylidene fluoride membranes were rinsed in 10 mM Tris-Cl, pH 7.2, 100 mM NaCl, and 0.1% Tween 20 (TBS-T) and blocked for 1 h at room temperature using 5% blocking solution (GE Healthcare) prepared in TBS-T. The membrane was incubated with indicated antibodies overnight (12–14 h) at 4°C followed by three washes with TBS-T.

The membrane was then incubated with anti-mouse IgG (at 1:6,000 dilution) or anti-rabbit IgG (at 1:30,000 dilution) conjugated to horseradish peroxidase for 1 h at room temperature, followed by three washes with TBS-T. Immunoreactive bands were visualized by chemiluminescence detected using Immobilon reagent (Millipore) on a Chemidoc XRS+ (Bio-Rad) imaging system. Anti-villin and anti-Na+/K+ adenosine triphosphatase antibodies were used at a dilution of 1:5,000.

Anti-pSTAT1 Ser 727, anti-pSTAT1 Tyr701, anti-STAT1, anti-TAP1, anti-ISG15, and anti–β-actin antibodies were used at a dilution of 1:1,000. Anti-Ido1 antibody was used at a dilution of 1:4,000. Anti-NHE3 antibody was used at a dilution of 1:2,000.

Anti-PKGII antibody was used at a dilution of 1:2,000. The sources of all commercial antibodies are shown in Table S2. GCC:4B11 monoclonal antibody was raised against the kinase homology domain of human GC-C and is available in the laboratory.

The supernatant from cultured cells was used for Western Blot analysis at a dilution of 1:100. The frequency of bowel movements was determined as previously described (De Palma et al., 2015). 4-wk-old mice were used, and the experiment was performed between 8:00 a.m.

And 9:00 a.m. Mice were placed in fresh autoclaved cages without any bedding material, and the number of pellets passed in the first 10 min was recorded as the frequency of bowel movements. Experiments were performed three times over the course of a year on independent litters.

To determine total gut transit time, nonfasted mice (7–8 wk old) were gavaged with 200 µl of nonabsorbable marker dye (6% wt/vol of carmine red in filter-sterilized 0.5% methylcellulose). The mice were housed individually in clean cages without bedding material with access to food and water. The mice were checked for output at 15-min intervals.

The time taken for the first fecal pellet with carmine red to be passed was recorded as the total gut transit time. Experiments were performed three times over the course of a year on independent litters. Small intestinal transit rate and GE were estimated using the GC and GE parameters as previously described (Sobczak et al., 2014) with a few modifications.

Briefly, 8–12-wk-old mice were fasted overnight with free access to water. On the day of the experiment, mice were weighed and orally gavaged with 200 µl of a filter-sterilized marker dye mixture (50 mg/100 ml phenol red in 0.5% methylcellulose). 30 min after marker dye administration, mice were sacrificed, the gastrointestinal tract was isolated and kept chilled to reduce further peristalsis, and dissection was conducted as fast as possible.

For the GC analysis, the small intestine was measured and divided into 10 equal parts. The intestinal segments were transferred to a tube containing 1 ml of distilled water and homogenized such that the intestinal contents were released into the water. The tubes were vortexed gently and centrifuged at 3,000 g for 5 min.

Following this, 250 µl of the supernatant was transferred to another fresh tube containing 250 µl of 1N NaOH, and color was allowed to develop. The intensity of color was calorimetrically measured at 560 nm using a spectrophotometer (Tecan Infinite M200 Pro. Tecan Switzerland).

GC of the small intestine was calculated using the following formula:GC = Σ [(% A per segment×segment number)/100],with GC ranges from 1 (minimum motility) to 10 (maximum motility). To determine the GE of mice, the stomach was dissected carefully, its contents were transferred to a tube containing 2 ml distilled water, and the mixture was vortexed gently followed by centrifugation at 3,000 g for 5 min. 500 µl of supernatant was transferred to another tube containing 500 µl of 1N NaOH to develop a maximum intensity of color.

The intensity of color (200 µl) was measured at 560 nm using a spectrophotometer. The percentage of dye that was present in the small intestine out of the total dye present in the stomach and the small intestine was a reflection of GE. To test linaclotide-mediated enhancement in the small bowel transit rate, GC and GE analyses were performed using the protocol described earlier (Bryant et al., 2010) with a few modifications.

The mice were fasted overnight with unlimited access to water. Mice were weighed and administered 100 µg/kg of body weight linaclotide (Cayman Chemicals) prepared in 25 mM Tris-HCl, pH 7.5, orally. Mice were replaced in their cages for 10 min and then sacrificed, and GC and GE analyses were performed.

To determine the effect of Cftr(inh)-172 (Sigma-Aldrich) on small bowel transit rate, overnight fasted mice were orally gavaged with 200 µg of Cftr(inh)-172 prepared in 10% wt/vol D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS). Control mice received the vehicle alone (Thiagarajah et al., 2004). Mice were returned to their cages for 3 h, after which GC and GE analyses were performed.

To determine the effect of the tenapanor (NHE3 inhibitor. MedChem Express) on small bowel motility, mice were fasted overnight and orally gavaged with 1 mg/kg tenapanor (McHugh et al., 2018) prepared in 10% TPGS. Control mice received the vehicle alone.

Mice were returned to their cages for 2 h, after which the GC and GE analyses were performed. Extraction of DNA from the fecal pellets of wild type (n = 11. 8 female and 3 male) and S839I (n = 10.

7 female and 3 male) mice was performed using the Fast DNA SPIN kit for soil (MP Biomedicals) according to the manufacturer’s protocol, with four bead-beating periods of 40 s. DNA concentration was normalized to 10 ng/µl by dilution with DNA elution solution (MP Biomedicals) to produce a final volume of 20 µl. DNA samples were sent to Clevergene Biocorp Pvt Ltd for PCR amplification of V3-V4 regions of 16S rRNA genes and paired-end sequencing (2 × 300 bp) on the Illumina MiSeq platform.

The V3-V4 hypervariable regions of the 16S rRNA genes were amplified using the 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) primers (Klindworth et al., 2013). The raw paired-end reads were obtained in fastq format from the Illumina MiSeq platform. Taxonomic abundance tables were generated from fastq files using the dada2 pipeline (v1.14.0) for paired-end reads (Callahan et al., 2016) in R (v3.6.1).

Briefly, quality score plots of sequence reads were inspected to determine the drop-off points in quality of reads based on which of the forward and reverse reads were truncated at 270 and 230 positions, respectively. Primer sequences were removed by trimming the 22 bp from the left end of the raw reads. Chimeric sequences were removed by using the remove Chimera Denovo function with the pooled sample inference method.

Taxonomy was assigned to the chimera-free amplicon sequence variants (ASVs) using Silva database (v138, downloaded from McLaren, 2020). Data were filtered to remove ASVs assigned as mitochondria, chloroplasts, or other nonbacterial kingdoms and ASVs with less than two frequencies in total (singletons) using the phyloseq R package (McMurdie and Holmes, 2013. V1.30.0).

Beta diversity measures were visualized using the principal coordinate analysis plot based on Bray-Curtis dissimilarity using the microbiome R package (Lahti et al., 2017. V1.8.0). Relative abundance at taxonomic levels of phyla and genera were based on ASV counts normalized as percentages (100 * [×/sum(×)]).

Significance of differences between Shannon diversity index was determined using Wilcoxon rank sum test (P <. 0.05) in the microbiome R package, while significant difference in relative abundance of taxa between wild type and S839I mice was tested using the Kruskal–Wallis test in STAMP statistical software (Parks et al., 2014). Prediction of the functional content of gut microbiome from 16S rRNA gene ASV count dataset and representative sequence of each ASV was performed using the Piphillin tool (Narayan et al., 2020).

In brief, this tool predicts functional attributes of microbial assemblages via direct nearest-neighbor matching between 16S rRNA gene amplicons and genomes from reference databases. Prediction was executed at 97% ID cutoff using KEGG (May 2020 release) and BioCyc22.5 reference databases. The output from Piphillin was analyzed by STAMP statistical software, using nonparametric Kruskal–Wallis test with Tukey-Kramer post hoc test (Parks et al., 2014).

This paper is dedicated to the memory of Dr. Torunn Fiskerstrand, whose enthusiasm for development of this mouse model was motivating. We thank Dr.

Halvor Sommerfelt for his interest and support for this work and Dr. Avinash R. Shenoy for careful reading of the paper and critical suggestions.

We acknowledge the help of Dr. Harini Ramani and Yashika Bopanna in the mouse experiments. Support from the Department of Biotechnology, Ministry of Science and Technology, India is acknowledged (BT/PR15216/COE/34/02/2017), as well as from DBT-IISc Partnership Program Phase-II (BT/PR27952/INF/22/212/2018/21.01.2019).

S.S. Visweswariah is a JC Bose National Fellow (SB/S2/JCB-18/2013) and a Margdarshi Fellow supported by the Wellcome Trust DBT India Alliance (IA/M/16/502606). Financial support from Helse Vest Norway, the Center for International Health, Department of Global Health and Primary Care, University of Bergen, Norway, and the Enteric treatment Initiative of PATH (https://www.path.org) is acknowledged toward the generation of the mouse model.

S.S. Visweswariah also acknowledges support from the Royal Society, UK, for a Collaborative Grant for Research Professors (IC60080), and funding from the Bill and Melinda Gates Grand Challenges Exploration Grant with Grant ID OPP1106646. Author contributions.

Chaukimath, M.M. Reshi, and S. Srinivas performed experiments and analyzed data.

A. Barman maintained and assisted in animal experimentation. V.

Banerjee wrote initial drafts of the manuscript. And S.S. Visweswariah conceived of the study, designed the analyses, and finalized the manuscript.Citation Claire Pujol, Anne Legrand, Livia Parodi, Priscilla Thomas, Fanny Mochel, Dario Saracino, Giulia Coarelli, Marijana Croon, Milica Popovic, Manon Valet, Nicolas Villain, Shahira Elshafie, Mahmoud Issa, Stephane Zuily, Mathilde Renaud, Cécilia Marelli-Tosi, Marine Legendre, Aurélien Trimouille, Isabelle Kemlin, Sophie Mathieu, Joseph G.

Gleeson, Foudil Lamari, Daniele Galatolo, Rana Alkouri, Chantal Tse, Diana Rodriguez, Claire Ewenczyk, Florence Fellmann, Thierry Kuntzer, Emilie Blond, Khalid H. El Hachimi, Frédéric Darios, Alexandre Seyer, Anastasia D. Gazi, Patrick Giavalisco, Silvina Perin, Jean-Luc Boucher, Laurent Le Corre, Filippo M.

Santorelli, Cyril Goizet, Maha S. Zaki, Serge Picaud, Arnaud Mourier, Sophie Marie Steculorum, Cyril Mignot, Alexandra Durr, Aleksandra Trifunovic, Giovanni Stevanin. Implication of folate deficiency in CYP2U1 loss of function.

J Exp Med 1 November 2021. 218 (11). E20210846.

Doi. Https://doi.org/10.1084/jem.20210846 Download citation file:.

What side effects may I notice from Lasix?

Side effects that you should report to your doctor or health care professional as soon as possible:

• blood in urine or stools
• dry mouth
• fever or chills
• hearing loss or ringing in the ears
• irregular heartbeat
• muscle pain or weakness, cramps
• skin rash
• stomach upset, pain, or nausea
• tingling or numbness in the hands or feet
• unusually weak or tired
• vomiting or diarrhea
• yellowing of the eyes or skin

Side effects that usually do not require medical attention (report to your doctor or health care professional if they continue or are bothersome):

• headache
• loss of appetite
• unusual bleeding or bruising

This list may not describe all possible side effects.

Get lasix prescription

Interim order respecting the importation, sale and advertising of drugs for use in relation to hypertension medications (September 16, 2020) and interim get lasix prescription order respecting the importation and sale of medical devices for use in relation to hypertension medications(March 18, 2020)The public release of safety and efficacy/effectiveness information reviewed under the 2 interim orders look what i found is governed by common law. Information requested for release is assessed case by case to determine what is CBI. Personal information is removed before the safety and efficacy/effectiveness information is released to the public.Following Health Canada’s review of an application, safety and efficacy information will be released as follows. Automatically disclosed in applications submitted under the interim order for importing, selling and advertising drugs (proactive release) disclosed on request in applications submitted under the interim order for importing and selling medical devices (released upon request)Information in applications that get lasix prescription have been authorized, including those authorized and then revoked, is in scope for public release.

This includes. Original application documents documents filed after market authorization is issued (filed at Health Canada’s request or to meet a condition of approval)Information in applications that are refused and were never authorized is out of scope for public release. This document does not apply to clinical information submitted to support the market authorization of a medical device under the Medical Device get lasix prescription Regulations or of a new drug submission under the Food and Drug Regulations (FDR). The exception are new drug submissions for hypertension medications indications submitted under the FDR.

For more information on the public release of this information, see the Public Release of Clinical Information. Guidance document.Also not applicable under this document is the CBI disclosure authority get lasix prescription under section 21.1(3)(c) of the Food and Drugs Act. This section permits the Minister of Health to disclose CBI to certain persons for the purpose of protection or promotion of human health or the safety of the public. For information on this authority, see the guidance document Disclosure of Confidential Business Information under Paragraph 21.1(3)(c) of the Food and Drugs Act.Proactive release of drug application informationWe will proactively publish safety and efficacy information used to support interim order drug applications upon authorization.

This includes clinical information in applications submitted under sections 3, 6 and 14 of the interim order.How to request clinical information in medical device applicationsWe will publish safety and effectiveness information used to support interim order medical device applications when we receive a request from the get lasix prescription public and within the limits of our administrative capacity. Requests made for multiple applications will be processed in sequence and subject to prioritization. Further prioritization may be given to products that have a greater impact on the health system, such as. Products that get lasix prescription are used a lot products that have a higher public interestRequests received for information in applications under the interim order will be prioritized over requests for clinical information in non-hypertension medications19-related drugs submissions and device applications.To request clinical information on medical device applications, use our special portal to submit an electronic request form.

Be sure to identify the product name listed on the following sites. Publication process Publication of safety and efficacy information used to support drug interim order applications The publication of information follows the process described in section 4 and Appendix C of the Public Release of Clinical Information guidance document.In accordance with PRCI timelines, we aim to publish a final redacted and anonymized package on our clinical information portal within 120 calendar days from starting the process. The process starts automatically on the day get lasix prescription an authorization is issued.Step 1. Notice to the company and request for proposed CBI redactions and anonymizationFollowing the authorization of a drug under the interim order, Health Canada will give the manufacturer an opportunity to take part in a process initiation meeting.

The first 60 days of the 120-day publication process is allocated for the company to review the clinical information. The company uses the Proposed Redaction Control Sheet (Appendix E, Public Release of Clinical get lasix prescription Information (PRCI) guidance document) to propose any redaction of CBI. Proposed CBI redactions should pertain to information that meets the definition of confidential business information. This is defined in Section 2 of the Food and Drugs Act, which mirrors common law in the context of confidential business information that meets each of the following 3 elements of the definition.

That is not publicly available in respect of which the person has taken measures that are reasonable in the circumstances to ensure that it remains not publicly available and that has actual get lasix prescription or potential economic value to the person or their competitors because it is not publicly available and its disclosure would result in a material financial loss to the person or a material financial gain to their competitorsFollowing an assessment of the proposals, text within an in-scope document found to meet the above definition will be protected. Similar to Public Release of Clinical Information policies, any information that meets the definition of “clinical information” will not be considered confidential business information. Exceptions to the PRCI regulations described in C.08.009.2(2)(a) and (b) of the Food and Drug Regulations or section 43.12(2)(a) and (b) of the Medical Device Regulations will be considered when applying redactions to confidential business information. Further information on the application of these exceptions can be found in get lasix prescription the Health Canada PRCI guidance document.All personal information should be anonymized in accordance with section 6 of the Public Release of Clinical Information guidance document.

The proposal package from the manufacturer should include. The proposed redaction control sheet the draft anonymization report annotated documentsManufacturers submit for Health Canada assessment using either CanadaPost ePost Connect or a suitable secure file transfer site of the manufacturer’s choosing.Step 2. Health Canada assessment get lasix prescription of company representationsWithin 30 days of receiving the proposal package, Health Canada will complete and return our assessment of the proposed CBI redactions and anonymization methodology. Proposed redactions that meet the definition of confidential business information will be protected.

We will review the anonymization methodology to ensure all personal information is protected while maximizing the disclosure of useful clinical information. Step 3 get lasix prescription. Revision of proposed CBI redactions and anonymizationIf proposed CBI redactions are rejected or revision is required to the anonymization methodology, in accordance with the Public Release of Clinical Information. Guidance document, the manufacturer will be given 15 days to make the revisions and resubmit.

We will send get lasix prescription our final assessment to the manufacturer within 5 days of receiving the revised package. Step 4. Finalization and publicationWithin 5 days of receiving our final assessment, the manufacturer must format and submit the final redacted and anonymization clinical documents within 5 days of receiving our final assessment. The final documents must comply get lasix prescription with the Guidance Document.

Preparation of Regulatory Activities using the Electronic Common Technical Document (eCTD) Format. These documents are to be submitted using the Common Electronic Submission Gateway. We will publish the final redacted documents within 5 days of receiving get lasix prescription the final sequence.Publication of safety and effectiveness information used to support medical device interim order applicationsThe publication of information within an interim order application will proceed through the abbreviated process described below. Our goal is to publish a final redacted and anonymized package on our clinical information portal within 120 calendar days from initiation of the process.Step 1.

Health Canada screening of requestsAfter we receive a request for information, we will retrieve the interim order application from docubridge (or other location). Information related to safety and effectiveness will be considered in-scope of get lasix prescription publication. Other information will not be released publicly. Only information available at the time the request is made will be considered for disclosure.

Information submitted after the original get lasix prescription request for disclosure will be considered for public release upon receipt of a subsequent request.Examples of in scope information include. Clinical testing information validation testing that supports the effectiveness of the product, including testing performed in vitro or in silico summaries or overviews on safety or efficacy pre- or post-market, including literature reviewsExamples of out of scope information include. Manufacturing details not related to safety or efficacy engineering and design details general documents, such as user manuals, package inserts and instructions for use individual patient information, such as patient listings and case report forms, that require extensive anonymization interim clinical study data (see the PRCI guidance)Step 2a. Health Canada assessment of confidential business information To reduce administrative burden get lasix prescription on the manufacturer, we will review in-scope records for confidential business information, as defined in Section 2 of the Food and Drugs Act, which mirrors common law in the context of confidential business information that meets each of the following 3 elements of the definition will be protected.

That is not publicly available in respect of which the person has taken measures that are reasonable in the circumstances to ensure that it remains not publicly available and that has actual or potential economic value to the person or their competitors because it is not publicly available and its disclosure would result in a material financial loss to the person or a material financial gain to their competitorsText in an in-scope document found to meet this definition will be redacted using a PDF redaction tool. Similar to Public Release of Clinical Information policies, any information that meets the definition of “clinical information” will not be considered confidential business information. Exceptions to the PRCI regulations are outlined section get lasix prescription 43.12(2)(a) and (b) of the Medical Device Regulations. These exceptions will be considered when applying redactions to confidential business information.

Further information on the application of these exceptions can be found in the PRCI guidance document.Step 2b. Assessing personal informationIn general, in-scope records do not contain a large volume of get lasix prescription personal identification information. Any personal information, as defined in the Privacy Act and in accordance with PRCI guidance, information that could help to identify an individual will be protected. For example, this can include the names of authors and investigators as well as subject identification numbers.A large volume of indirectly identifying information is not expected in the medical device records that are in-scope of publication.

Consequently, limited protection of personal get lasix prescription information is anticipated.Personal information will be redacted using a PDF redaction tool. Step 3. Notice to the company and request for redaction proposalFollowing the review and redaction of in scope documents, we will send the manufacturer a written notice indicating our intent to publish the identified documents. A copy of the release package will be sent for the manufacturer’s review get lasix prescription.

Any further proposed redactions by the manufacturer must be received within 14 calendar days.Manufacturer are asked to use the Proposed Redaction Control Sheet (see Appendix E of the PRCI guidance document) to suggest further redactions.Step 4. Health Canada assessment of company representationsAny further redactions proposed by the manufacturer will be assessed in accordance with the process outlined in step 2, above. Those that meet the definition get lasix prescription of personal or confidential business information will be accepted.Step 5. PublicationIn-scope documents will be published within 120 days following receipt of the request.

The redacted information will be uploaded to the Clinical Information Portal, indexed by application number. Published documents will carry get lasix prescription a watermark and be subject to terms of use, as described in the PRCI guidance.Mailing addressInformation Science and Openness DivisionResource Management and Operations DirectorateHealth Products and Food BranchHealth Canada Graham Spry Building 250 Lanark Ave Ottawa ON K1A 0K9 Telephone. 613-960-4687Email. Hc.clinicaldata-donneescliniques.sc@canada.ca Terminology and definitions Anonymization.

Means the process through which personal information is modified get lasix prescription by. removing direct identifiers and any related code that would enable linkage with identifying information and ensuring that the remaining indirect identifiers no longer present a serious possibility of re-identifying an individual CBI. Confidential business information, as meant in common law and as defined in Section 2 of the Food and Drugs Act. in respect of a person to whose business get lasix prescription or affairs the information relates, means (subject to the regulations) business information that.

Is not publicly available in respect of which the person has taken measures that are reasonable in the circumstances to ensure that it remains not publicly available has actual or potential economic value to the person or their competitors because it is not publicly available and its disclosure would result in a material financial loss to the person or a material financial gain to their competitors Clinical information. Means information in respect of a clinical trial, clinical studies or investigational testing, such as. clinical overviews, clinical get lasix prescription summaries and clinical study reports for drugs summaries and detailed information of all clinical studies and investigational testing that provided evidence of safety and effectiveness for medical devices Clinical study report. Means an "integrated" full report of an individual study of any therapeutic, prophylactic or diagnostic agent (drug or treatment) conducted in patients, in which.

the clinical and statistical description, presentations and analyses are integrated into a single report incorporating tables and figures into the main text of the report or at the end of the text appendices contain the protocol, sample case report forms, investigator-related information, information related to the test drugs/investigational products, including active control/comparators, technical statistical documentation, related publications, patient data listings and technical statistical details such as derivations, computations, analyses and computer output FDA. Food and Drugs Act get lasix prescription FDR. Food and Drug Regulations IMDRF ToC. International Medical Device Regulators Forum Table of Contents Medical device.

Has the same meaning as get lasix prescription insee the Medical Devices Regulations. For information on the classification of medical devices, please see the guidance documents on the. risk-based classification system for in vitro diagnostic devices (IVDDs) risk-based classification system for non-in vitro diagnostic devices (non-IVDDs) Non-commercial purpose. Means the information will not be used to support a marketing authorization application anywhere in the get lasix prescription world or sold or traded to another person Personal information.

Has the same meaning as in Section 3 of the Privacy Act Related linksOn this page About the guidance document This guidance document supports the Interim Order Respecting Drugs, Medical Devices and Foods for a Special Dietary Purpose in Relation to hypertension medications. The Minister of Health approved the Interim Order on March 30, 2020, to address the unprecedented demand and urgent need for medical devices to treat, diagnose and protect Canadians against hypertension medications. The guidance covers sections 15 to 19 of get lasix prescription the Interim Order. It remains in effect as long as the Interim Order is in effect.

Under the Interim Order, manufacturers and importers must report medical device shortages related to hypertension medications to Health Canada. The devices to get lasix prescription which the shortages apply are on the List of Medical Devices — Notification of Shortages (specified medical devices). A specified medical device is a device that is either. set out in the list of medical devices or part of a category of medical devices that is set out in that list The guidance is intended to help manufacturers and importers meet their regulatory obligations.

It outlines get lasix prescription their responsibilities concerning the mandatory reporting of medical device shortages. About medical device shortages and reporting A medical device shortage occurs when a manufacturer is unable to meet Canadian market demand for the device or for its components, accessories, parts or consumable materials. This does not apply when a substitute device, component, accessory, part or consumable material is available in Canada. There are get lasix prescription 2 types of shortages.

actual, when the current supply can’t meet current demand anticipated, when the future supply can’t meet projected demand Manufacturers and importers must. report a medical device shortage provide a shortage status update if there is a change in the shortage information submitted provide additional information related to a shortage when requested by Health Canada report an end of a medical device shortage This guidance document also provides some guidance on how to voluntarily report a medical device shortage that does not fall under the Interim Order. Everyone has a role to play Manufacturers and importers Manufacturers and importers have a key role to play in preventing and reducing get lasix prescription the impact of medical device shortages. They can control the volume of medical devices in the supply chain and can take steps to resolve a medical device shortage when one occurs.

They are also in the best position to communicate to customers about the availability of their devices. When a manufacturer experiences a shortage of a critical medical device it sells, we expect that the manufacturer will take all necessary measures to resolve the shortage as quickly as possible.

procedures when generic lasix online releasing information types of information that fall under the guidelines for CBI and that may be eligible for redaction protection of personal informationScope and application http://solarhairsalon.com/?p=1641 This document applies to information relied upon to issue a market authorization under the. Interim order respecting the importation, sale and advertising of drugs for use in relation to hypertension medications (September 16, 2020) and interim order respecting the importation and sale of medical devices for use in relation to hypertension medications(March 18, 2020)The public release of safety and efficacy/effectiveness information reviewed under the 2 interim orders is governed by common law. Information requested for release is assessed case by case to determine what is CBI. Personal information generic lasix online is removed before the safety and efficacy/effectiveness information is released to the public.Following Health Canada’s review of an application, safety and efficacy information will be released as follows.

Automatically disclosed in applications submitted under the interim order for importing, selling and advertising drugs (proactive release) disclosed on request in applications submitted under the interim order for importing and selling medical devices (released upon request)Information in applications that have been authorized, including those authorized and then revoked, is in scope for public release. This includes. Original application documents documents filed after market authorization is issued (filed at Health Canada’s request or to meet a condition of generic lasix online approval)Information in applications that are refused and were never authorized is out of scope for public release. This document does not apply to clinical information submitted to support the market authorization of a medical device under the Medical Device Regulations or of a new drug submission under the Food and Drug Regulations (FDR).

The exception are new drug submissions for hypertension medications indications submitted under the FDR. For more information on the public release of this information, see the generic lasix online Public Release of Clinical Information. Guidance document.Also not applicable under this document is the CBI disclosure authority under section 21.1(3)(c) of the Food and Drugs Act. This section permits the Minister of Health to disclose CBI to certain persons for the purpose of protection or promotion of human health or the safety of the public.

For information on this authority, see the guidance document Disclosure of Confidential Business Information under Paragraph 21.1(3)(c) of the generic lasix online Food and Drugs Act.Proactive release of drug application informationWe will proactively publish safety and efficacy information used to support interim order drug applications upon authorization. This includes clinical information in applications submitted under sections 3, 6 and 14 of the interim order.How to request clinical information in medical device applicationsWe will publish safety and effectiveness information used to support interim order medical device applications when we receive a request from the public and within the limits of our administrative capacity. Requests made for multiple applications will be processed in sequence and subject to prioritization. Further prioritization may be given to products that have a greater impact on the health system, generic lasix online such as.

Products that are used a lot products that have a higher public interestRequests received for information in applications under the interim order will be prioritized over requests for clinical information in non-hypertension medications19-related drugs submissions and device applications.To request clinical information on medical device applications, use our special portal to submit an electronic request form. Be sure to identify the product name listed on the following sites. Publication process Publication of safety and efficacy information used to support drug interim order applications The publication of information follows generic lasix online the process described in section 4 and Appendix C of the Public Release of Clinical Information guidance document.In accordance with PRCI timelines, we aim to publish a final redacted and anonymized package on our clinical information portal within 120 calendar days from starting the process. The process starts automatically on the day an authorization is issued.Step 1.

Notice to the company and request for proposed CBI redactions and anonymizationFollowing the authorization of a drug under the interim order, Health Canada will give the manufacturer an opportunity to take part in a process initiation meeting. The first 60 days of the 120-day publication process is allocated generic lasix online for the company to review the clinical information. The company uses the Proposed Redaction Control Sheet (Appendix E, Public Release of Clinical Information (PRCI) guidance document) to propose any redaction of CBI. Proposed CBI redactions should pertain to information that meets the definition of confidential business information.

This is defined in Section 2 of the generic lasix online Food and Drugs Act, which mirrors common law in the context of confidential business information that meets each of the following 3 elements of the definition. That is not publicly available in respect of which the person has taken measures that are reasonable in the circumstances to ensure that it remains not publicly available and that has actual or potential economic value to the person or their competitors because it is not publicly available and its disclosure would result in a material financial loss to the person or a material financial gain to their competitorsFollowing an assessment of the proposals, text within an in-scope document found to meet the above definition will be protected. Similar to Public Release of Clinical Information policies, any information that meets the definition of “clinical information” will not be considered confidential business information. Exceptions to the PRCI regulations described in C.08.009.2(2)(a) and (b) of the Food and Drug Regulations or section 43.12(2)(a) and (b) of the Medical Device Regulations generic lasix online will be considered when applying redactions to confidential business information.

Further information on the application of these exceptions can be found in the Health Canada PRCI guidance document.All personal information should be anonymized in accordance with section 6 of the Public Release of Clinical Information guidance document. The proposal package from the manufacturer should include. The proposed redaction control generic lasix online sheet the draft anonymization report annotated documentsManufacturers submit for Health Canada assessment using either CanadaPost ePost Connect or a suitable secure file transfer site of the manufacturer’s choosing.Step 2. Health Canada assessment of company representationsWithin 30 days of receiving the proposal package, Health Canada will complete and return our assessment of the proposed CBI redactions and anonymization methodology.

Proposed redactions that meet the definition of confidential business information will be protected. We will review the anonymization methodology to ensure all personal information is protected while maximizing generic lasix online the disclosure of useful clinical information. Step 3. Revision of proposed CBI redactions and anonymizationIf proposed CBI redactions are rejected or revision is required to the anonymization methodology, in accordance with the Public Release of Clinical Information.

Guidance document, the manufacturer will be given 15 days to make the revisions generic lasix online and resubmit. We will send our final assessment to the manufacturer within 5 days of receiving the revised package. Step 4. Finalization and publicationWithin 5 days generic lasix online of receiving our final assessment, the manufacturer must format and submit the final redacted and anonymization clinical documents within 5 days of receiving our final assessment.

The final documents must comply with the Guidance Document. Preparation of Regulatory Activities using the Electronic Common Technical Document (eCTD) Format. These documents generic lasix online are to be submitted using the Common Electronic Submission Gateway. We will publish the final redacted documents within 5 days of receiving the final sequence.Publication of safety and effectiveness information used to support medical device interim order applicationsThe publication of information within an interim order application will proceed through the abbreviated process described below.

Our goal is to publish a final redacted and anonymized package on our clinical information portal within 120 calendar days from initiation of the process.Step 1. Health Canada screening of requestsAfter we generic lasix online receive a request for information, we will retrieve the interim order application from docubridge (or other location). Information related to safety and effectiveness will be considered in-scope of publication. Other information will not be released publicly.

Only information available at the time the request is generic lasix online made will be considered for disclosure. Information submitted after the original request for disclosure will be considered for public release upon receipt of a subsequent request.Examples of in scope information include. Clinical testing information validation testing that supports the effectiveness of the product, including testing performed in vitro or in silico summaries or overviews on safety or efficacy pre- or post-market, including literature reviewsExamples of out of scope information include. Manufacturing details not related generic lasix online to safety or efficacy engineering and design details general documents, such as user manuals, package inserts and instructions for use individual patient information, such as patient listings and case report forms, that require extensive anonymization interim clinical study data (see the PRCI guidance)Step 2a.

Health Canada assessment of confidential business information To reduce administrative burden on the manufacturer, we will review in-scope records for confidential business information, as defined in Section 2 of the Food and Drugs Act, which mirrors common law in the context of confidential business information that meets each of the following 3 elements of the definition will be protected. That is not publicly available in respect of which the person has taken measures that are reasonable in the circumstances to ensure that it remains not publicly available and that has actual or potential economic value to the person or their competitors because it is not publicly available and its disclosure would result in a material financial loss to the person or a material financial gain to their competitorsText in an in-scope document found to meet this definition will be redacted using a http://www.em-holtzheim.site.ac-strasbourg.fr/2021/01/25/carnaval/ PDF redaction tool. Similar to Public Release of Clinical Information policies, any information that meets the definition generic lasix online of “clinical information” will not be considered confidential business information. Exceptions to the PRCI regulations are outlined section 43.12(2)(a) and (b) of the Medical Device Regulations.

These exceptions will be considered when applying redactions to confidential business information. Further information on the application of these exceptions generic lasix online can be found in the PRCI guidance document.Step 2b. Assessing personal informationIn general, in-scope records do not contain a large volume of personal identification information. Any personal information, as defined in the Privacy Act and in accordance with PRCI guidance, information that could help to identify an individual will be protected.

For example, this can generic lasix online include the names of authors and investigators as well as subject identification numbers.A large volume of indirectly identifying information is not expected in the medical device records that are in-scope of publication. Consequently, limited protection of personal information is anticipated.Personal information will be redacted using a PDF redaction tool. Step 3. Notice to the company generic lasix online and request for redaction proposalFollowing the review and redaction of in scope documents, we will send the manufacturer a written notice indicating our intent to publish the identified documents.

A copy of the release package will be sent for the manufacturer’s review. Any further proposed redactions by the manufacturer must be received within 14 calendar days.Manufacturer are asked to use the Proposed Redaction Control Sheet (see Appendix E of the PRCI guidance document) to suggest further redactions.Step 4. Health Canada generic lasix online assessment of company representationsAny further redactions proposed by the manufacturer will be assessed in accordance with the process outlined in step 2, above. Those that meet the definition of personal or confidential business information will be accepted.Step 5.

PublicationIn-scope documents will be published within 120 days following receipt of the request. The redacted information will generic lasix online be uploaded to the Clinical Information Portal, indexed by application number. Published documents will carry a watermark and be subject to terms of use, as described in the PRCI guidance.Mailing addressInformation Science and Openness DivisionResource Management and Operations DirectorateHealth Products and Food BranchHealth Canada Graham Spry Building 250 Lanark Ave Ottawa ON K1A 0K9 Telephone. 613-960-4687Email.

Hc.clinicaldata-donneescliniques.sc@canada.ca Terminology and definitions Anonymization generic lasix online. Means the process through which personal information is modified by. removing direct identifiers and any related code that would enable linkage with identifying information and ensuring that the remaining indirect identifiers no longer present a serious possibility of re-identifying an individual CBI. Confidential business information, as meant in common law and generic lasix online as defined in Section 2 of the Food and Drugs Act.

in respect of a person to whose business or affairs the information relates, means (subject to the regulations) business information that. Is not publicly available in respect of which the person has taken measures that are reasonable in the circumstances to ensure that it remains not publicly available has actual or potential economic value to the person or their competitors because it is not publicly available and its disclosure would result in a material financial loss to the person or a material financial gain to their competitors Clinical information. Means information in respect of a clinical trial, clinical studies generic lasix online or investigational testing, such as. clinical overviews, clinical summaries and clinical study reports for drugs summaries and detailed information of all clinical studies and investigational testing that provided evidence of safety and effectiveness for medical devices Clinical study report.

Means an "integrated" full report of an individual study of any therapeutic, prophylactic or diagnostic agent (drug or treatment) conducted in patients, in which. the clinical and statistical description, presentations and analyses are integrated into a single report incorporating tables and generic lasix online figures into the main text of the report or at the end of the text appendices contain the protocol, sample case report forms, investigator-related information, information related to the test drugs/investigational products, including active control/comparators, technical statistical documentation, related publications, patient data listings and technical statistical details such as derivations, computations, analyses and computer output FDA. Food and Drugs Act FDR. Food and Drug Regulations IMDRF ToC.

International Medical Device generic lasix online Regulators Forum Table of Contents Medical device. Has the same meaning as insee the Medical Devices Regulations. For information on the classification of medical devices, please see the guidance documents on the. risk-based classification system for in vitro diagnostic devices (IVDDs) risk-based classification system for generic lasix online non-in vitro diagnostic devices (non-IVDDs) Non-commercial purpose.

Means the information will not be used to support a marketing authorization application anywhere in the world or sold or traded to another person Personal information. Has the same meaning as in Section 3 of the Privacy Act Related linksOn this page About the guidance document This guidance document supports the Interim Order Respecting Drugs, Medical Devices and Foods for a Special Dietary Purpose in Relation to hypertension medications. The Minister of Health approved the Interim Order on March 30, 2020, to address the unprecedented demand and urgent need for medical devices to generic lasix online treat, diagnose and protect Canadians against hypertension medications. The guidance covers sections 15 to 19 of the Interim Order.

It remains in effect as long as the Interim Order is in effect. Under the Interim Order, manufacturers and importers must report medical device generic lasix online shortages related to hypertension medications to Health Canada. The devices to which the shortages apply are on the List of Medical Devices — Notification of Shortages (specified medical devices). A specified medical device is a device that is either.

set out in the list of medical devices or part of a generic lasix online category of medical devices that is set out in that list The guidance is intended to help manufacturers and importers meet their regulatory obligations. It outlines their responsibilities concerning the mandatory reporting of medical device shortages. About medical device shortages and reporting A medical device shortage occurs when a manufacturer is unable to meet Canadian market demand for the device or for its components, accessories, parts or consumable materials. This does not generic lasix online apply when a substitute device, component, accessory, part or consumable material is available in Canada.

There are 2 types of shortages. actual, when the current supply can’t meet current demand anticipated, when the future supply can’t meet projected demand Manufacturers and importers must. report a medical device shortage provide a shortage status update if there is a change in the shortage information submitted provide additional information related to a shortage when requested by Health Canada report an end of a medical device shortage This guidance document also provides some guidance on how to voluntarily report a medical device shortage that does not fall under the Interim Order. Everyone has a role to play Manufacturers and importers Manufacturers and importers have a key role to play in preventing and reducing the impact of medical device shortages.

They can control the volume of medical devices in the supply chain and can take steps to resolve a medical device shortage when one occurs. They are also in the best position to communicate to customers about the availability of their devices.

Can lasix make you constipated

Medication errors have been a leading can lasix make you constipated low cost lasix cause of preventable harm for decades. Assiri and colleagues report that the cost of medication error worldwide exceeds $42 billion, or approximately 5%–6% of all hospitalisations.1 While this topic has been closely studied since its first appearance in scientific literature in 1953,2 the problems continue to evolve alongside changes to the medication-use system can lasix make you constipated. The medication-use system is a function of many elements. Widespread transitions from paper-based to electronic health records have affected drug ordering and prescribing, documentation, transcribing, dispensing, administering and monitoring in ways that challenge traditional approaches to reducing errors that predate electronic records.3 In addition, the introduction of over 7000 branded small molecules or biologics, generics and biosimilars that overlap numerous therapeutic areas increased dependence on specialty care for people with multiple chronic conditions, and navigating transitions throughout the range of primary to quaternary care have all complicated the ability of health systems to manage individual patient medication needs safely.4 Thus, solutions to address common medication errors 10 or 20 years ago may quickly become outdated in our fast-paced healthcare sector.Medication errors can either be intercepted prior to reaching can lasix make you constipated the patient or produce adverse drug events (ADEs) ranging from benign to life-threatening.

Concerning prevalence rates of ADEs in hospitalised patients have been reported at 3.22% in the UK, 4.78% in Germany and can lasix make you constipated 5.64% in the USA.5 For a country the size of the USA, the US Food and Drug Administration reports that this rate represents over 100 000 ADEs per year. However, these data relate only to the more severe ADEs. Those resulting in death, a life threatening health state, hospitalisation, disability or birth defect.6 These figures therefore encapsulate pain and suffering as captured in administrative data but do not include the multitude of patients who missed one or more days of can lasix make you constipated work or school, developed symptoms necessitating an outpatient or emergency room visit, induced long-term harm, or the attendant health system costs. The data therefore give only part of the overall picture.In contrast, based on a comprehensive analysis of UK data, the study by Elliott and colleagues in this issue attempts to illustrate the true full impact of medication errors and the associated risk of ADEs.7 Of the 237 million medication errors estimated to occur in England each year, 66 million are potentially clinically significant and result in 181 thousand hospital days and 1708 deaths at the cost of £98 million to the National Health Service.

However, the aetiology and factors influencing medication errors that lead can lasix make you constipated to these ADEs exceed ‘ubiquitous medicine use’ in the country. That is, the causes can lasix make you constipated of ADEs are multifaceted. In this case, comprehensive improvement of the medication-use system should not be overlooked—and its multifaceted nature is likely to require the execution of quality improvement initiatives across many domains.Elliott and colleagues break down medication errors by stage within the medication-use system to highlight the degree to which these issues are multifaceted. It comes as can lasix make you constipated little surprise that across primary care, secondary care and care homes, prescribing, dispensing, administration and monitoring errors are prominent.

However, the degree to which data are missing is also concerning and therefore may underestimate the prevalence and costs of medication errors. How can any health can lasix make you constipated system, let alone an entire National Health Service devise best practices to reduce medication errors when data that present a substantial proportion of variability in ADEs are missing?. ‘No UK data available’ in tables throughout Elliott and colleagues’ paper (ie, no comparable UK data were available for particular settings, such as care homes) is as insightful as can lasix make you constipated the numbers that are displayed since it presents an opportunity to improve quality of care informed by an investment in better data, among other needs.As with any quality improvement initiative, beginning with a framework to reduce ADEs as a result of medication error requires an established structure.8 The ‘five rights’ of medication administration offer health systems one potential structure on which to ensure individuals receive the right treatment to maximise clinical benefit and minimise harm. The right patient, the right drug, the right dose, the right route and the right time.9 Building from these principles, it becomes apparent that methods and technologies for interdiction of medication error and preventable ADEs are still being refined along with variability in execution.

Relatively simple solutions such as clear prescription labelling and safe packaging, multiple prescriber and pharmacy tracking to capture drug interaction risk, along with information sharing and advances in drug therapy stewardship, are examples of processes around which to build a quality improvement programme from the five rights structure that may achieve reduced rates of ADEs.4 Further targeting of these improvements within health system components where medication errors are most common, such as ambulatory and primary care settings and transitions of care, would represent efficient use of healthcare resources to reduce ADEs.1By addressing issues in primary care and outpatient settings, the healthcare sector would also minimise the number of ADEs that result in more expensive can lasix make you constipated secondary, tertiary and quaternary care, thereby increasing the probability of additional drug–drug interactions or other risks of medication errors. Further to this are settings and spaces where prescription practices are engaged, fulfilled and monitored. Providers and pharmacists rarely coexist in the same clinical settings in primary, outpatient and ambulatory care as they do in tertiary and quaternary care where the medical community has already recognised the importance of including pharmacists in patient rounds to review and reconcile medication errors.10 Past studies have noted that when the pharmacist is part of a clinical team to address patient needs within complex medication strategies, reductions in ADEs can be achieved throughout various healthcare settings.11–13 While the physically aligned presence of providers and pharmacists may not be as straightforward to facilitate in primary and outpatient care, increased can lasix make you constipated telecommunication throughout the medication use process, including computer order entry and medication reconciliation, could resolve issues that may otherwise lead to medication errors and subsequent ADEs.As the research of Elliott et al7 and other findings highlight, ADEs are a costly, harmful issue that remains prevalent in global healthcare. The added complexity created by layering healthcare delivery across can lasix make you constipated many settings of primary and specialty care creates gaps in communication where prescribers lack means or availability to actively communicate with pharmacists to identify and resolve potential medication errors.

The sheer increase in volumes of prescription medications that outpaces process efficiencies also challenges the ability of these two stakeholders to communicate directly on a per-patient basis. However, medication reviews focused on patients who take multiple prescriptions, have debilitating long-term conditions or have recently experienced acute decompensation that could make them particularly vulnerable to repeat episodes are an important focus for whom to narrow the degree of communication by default over medication review.14Beyond these suggestions for quality can lasix make you constipated improvement based on current information, the study by Elliott and colleagues highlights the need for additional data to further direct efforts towards efficient means of sustaining reduced ADE rates. Missing data are prevalent throughout the field of ADE outcomes, either because medication errors fail to meet the threshold that institutions such as the can lasix make you constipated US Food and Drug Administration set for a sentinel event or because such errors go completely unnoticed without being recorded as an episode within the health system. Many nations facing the reality of spending millions on ADEs could more proactively invest in improved reporting systems to precisely capture medication errors data, and which instances lead to minor as opposed to major ADEs, and the systems and clinical factors predicting them.

These investments in better and broader data collection and quality improvement can lasix make you constipated programme implementation often frighten away health system directors who fail to recognise the balance between action and reaction. Elliott and colleagues’ expected value of the economic burden of ADE is almost certainly an underestimate. If much of the data on ADEs are missing from the UK system, especially at transitions of care, and other ADEs go under-reported, then the current estimate of £98 million per annum is lower than the true medical and societal cost of this issue, can lasix make you constipated including non-monetary clinical disutility. The alternative cost scenario that Elliott and colleagues present in the range of £728 million per annum is perhaps a more realistic figure and one that justifies spending on quality improvement programming to offset hundreds of millions in avoidable costs.Thus, reporting systems that captures a wider range of ADEs, coupled with improved modes of communication between providers and pharmacists, as well can lasix make you constipated as a systematic effort to conduct root cause analysis that assist health systems to identify the nature of ADEs and evaluate potential solutions, are possibly cost-effective investments.15 The value of this information is imperative to inform more elaborate systems of medication management and target points of communication between providers and pharmacists to reconcile potential instances of medication error.16 Putting a learning health system model into place such as this—perhaps facilitated by machine learning—makes it more likely that damaging medication errors become more a part of our past history than an issue that the medical literature continues to review.For the past two decades, patient-centredness has served as one of six acknowledged dimensions of healthcare quality.1 Initially, healthcare institutions described patient centredness superficially—clean waiting rooms, hotel-like bed and board, access to innovative medical technology—and measured it with crude satisfaction scales.

The concept of patient-centred care evolved into a model attuned to the patient experience of care, defined by the interactions between patients and providers and the care environment.2 This patient experience model of patient-centred care has deep normative roots around principles of the patient as the locus of control and a demand for individualisation and customisation of care based on the patient rather than clinician.3 Empirically, patient experience is associated with health outcomes when defined and measured in a timely manner as a specific care experience or interaction between a patient and a healthcare provider.4 The importance of honouring the patient experience is now a widely appreciated construct and a common measure of healthcare quality with a deep evidence base.5 The Hospital Consumer Assessment of Healthcare Providers and Systems, Consumer Assessment of Health Providers and Systems Survey and Press Ganey patient satisfaction measures are ubiquitous measures of quality defining patient experiences of care.Moving beyond patient experience measuresThe effort to transform healthcare systems from clinician to patient centred is not complete. Honouring, measuring and ameliorating patients’ experiences of care is necessary but not sufficient and represents only the first stop on the journey to patient-centred care.6 The second stop is one that can lasix make you constipated nests the locus of control with patients and caregivers. Patients’ control over healthcare decisions is useful only when transparency exists in all aspects of care. Evidence, costs, processes, outcomes and errors.3 Unfortunately, claims that patients should have control and transparent understanding of all aspects of care have can lasix make you constipated largely been ignored due to institutional inertia, lack of financial incentives and the primacy of professionals.

In essence, there are few incentives to change this orientation, and clinicians too often perceive confrontation and frustration rather browse around here than partnership.7The primacy of physician professionalism stems from professional control over scientific knowledge and nurse professionalism from control over the practice environment, both bolstered by years of training and can lasix make you constipated experience. This professional model held for nearly a century when acute illnesses were the primary reason people sought medical care with the assumption that treatments were focused on cure (return to health) and/or alleviation of symptoms (removal of the disease).8 In contrast, healthcare in the 21st century primarily focuses on managing chronic diseases for which there are few cures. In the context of multiple chronic conditions (multimorbidity), the desired outcomes of healthcare are no longer obvious because they extend beyond the can lasix make you constipated goals of curing diseases or prolonging life. Multimorbidity also produces trade-offs among treatments, conditions and possible outcomes.9 For patients with multimorbidity, evidenced-based treatments are often lacking and, when present, there may be conflicts or incongruences across conditions.10 Effective management of chronic conditions requires active, ongoing participation by patients and caregivers outside of healthcare settings.

The intensity of can lasix make you constipated this management can be burdensome, further impacting patient experiences and even outcomes.11 Healthcare professionals now increasingly understand the need to share the burden of treatment decisions with their multimorbid patients.Patient centredness as healthcare that achieves patient prioritiesThe next stop on the journey to patient-centred care is the establishment of collaborative partnerships between healthcare professionals and patients.6 Productive partnerships require a medium for shared understanding that does not default to professional expertise and clinical practice guidelines. We have asserted that patient priorities are the necessary medium for focusing collaboration, discussions and healthcare decisions, especially in the context of complex, chronic illnesses.10 We precisely define patient priorities as the combination of the specific and realistic outcomes and activities (health outcome goals) that individuals want based on what matters most to them and the healthcare activities, including medications, self-care, tests and visits that they are willing and able to perform (healthcare preferences) to achieve their outcome goals.12 Evidence and professional judgement still guide which treatments are relevant, but clinicians should partner with their patients to select and adjust care based on a health goal as opposed to individual disease states.13 Pragmatic studies demonstrate that this patient priorities approach to care reduces polypharmacy and patient-reported treatment burden while increasing care that aligns with patient goals.14 15 Patients and clinicians describe this process as practical and beneficial.16Measuring goal attainment as a patient-centred care quality measureTo promote and disseminate patient priorities-aligned can lasix make you constipated care, novel quality measures are necessary. These quality metrics would evaluate the process for collaboratively identifying patient goals and care preferences and the degree to which patient goals are attained. In the can lasix make you constipated current issue of BMJ Quality and Safety, Giovannetti et al17 describe the results of an innovative study that evaluated the feasibility of two different approaches to developing quality measures of goals-based care.

The study assessed the implementation of these measures into diverse clinical settings and the subsequent interpretability and usefulness of the measures based on the data generated from either approach.As Giovannetti and colleagues describe, the key gap in evaluating goals-based care is the presence of measures for setting and documenting goals as well as tracking goal progress and attainment.17 In routine care, patient goals and care preferences are infrequently and haphazardly written and communicated, often conflicting, and typically focus on end-of-life care or chronic disease biomarkers.18–21 To address these gaps, the authors adapted goal attainment scaling, a reliable and valid approach for measuring goal setting and goal attainment in research studies.22 23 The authors asked patients and clinicians to jointly set a goal and define a set of possible outcomes along a five-point scale. They later discussed and then individually can lasix make you constipated rated the degree of goal attainment. The other approach evaluated by Giovannetti and colleagues17 can lasix make you constipated is the use of patient-reported outcome measures (PROMs), which are often used to measure specific domains (eg, mood, functioning, symptoms and so on) of health-related quality of life.24–26 In their study, Giovannetti et al17 asked patients and clinicians to jointly set a goal and then select a PROM that best matches that goal. At follow-up, the patient completed the same PROM to assess change over time.

Patients and clinicians were given a dozen PROMs from which to select.The study design and results of can lasix make you constipated the study by Giovannetti et al are both novel and provocative. The authors found that clinicians were more likely to implement goal attainment scaling, noted to can lasix make you constipated be practical to implement, compared with the PROM approach. Furthermore, clinicians found goal attainment scaling more useful for determining which services and supports to recommend and for helping patients achieve their goals. Contrary to common assumptions, the authors can lasix make you constipated found that clinicians and patients set goals collaboratively and focused on patient-centred outcomes rather than disease processes or biomarkers.

These findings suggest that implementation of a goals-based approach in routine care is feasible and demonstrate promise for fostering the shift from disease to patient-centred care.The lack of appeal for the PROM approach is surprising given their broad acceptance as quality measures.27 PROMs are effective tools for measuring particular behaviours, activities or symptoms that are either specific to a disease, such as diabetes,28 or reflect overall health-related quality of life.29 As quality metrics, PROMs provide patient-centred measures that can be applied across a population of patients, such as the Patient Health Questionnaire for measuring depression symptoms. However, patients and clinicians seem to prefer goals-based approaches, such as goal attainment scaling30 and patient priorities care,10 because they better reflect the goals can lasix make you constipated of specific individuals within the context of their own lives. We have shown that when older patients set goals that are specific to their individual lives, they typically fall into one of four can lasix make you constipated health-related values categories. (1) social and spiritual connections, (2) functioning and independence, (3) life enjoyment and pleasurable activities and (4) balancing quality and quantity of life (managing health).31 32 We have trained clinicians to identify specific and realistic goals based on what matters most to patients by initiating conversations around the four health values categories.12 These conversations can be efficiently incorporated in clinic visits and during telehealth encounters.

In another clinical trial, we demonstrated that a patient goals-based approach can significantly improve scores on a validated depression-specific PROM compared with routine guidelines-based care.33 These findings suggest that individualised approaches to goal attainment can be coupled with PROMs to provide a balanced can lasix make you constipated (individualised goals along with population-level measures) approach to quality measurement of patient-centred care.Financial incentives to promote patient-centred careTo facilitate dissemination of patient priorities aligned care, health insurers should support targeted financial incentives to facilitate widespread adoption into routine care. First, time-based reimbursement for clinical encounters with patients is vital. Medicare’s care management billing codes for annual wellness, advanced care planning can lasix make you constipated and chronic care management are also potential options. Establishment of novel value-based care management codes that are specific to priorities setting and measuring goal can lasix make you constipated progress and attainment would be key drivers of this effort.

Furthermore, these codes should support involvement of a range of health professionals. Training opportunities supported can lasix make you constipated by continuing education credits would further promote patient priorities care. Common concerns about quality measures focused on goal attainment include the setting of unrealistic or inappropriate goals, playing the system with easily attained goals and the nuances of patient–caregiver–clinician goal alignment. These are all can lasix make you constipated practical challenges to achieving a mature goals-aligned care process.

However, at this can lasix make you constipated early stage of development, Medicare should promote all efforts to implement value-based care management codes even if they are used primarily for financial incentives. Any impetus that encourages goal-based conversations and goal setting among patients, caregivers and clinicians will promote the necessary paradigm shift from guidelines-based care to goals-based care even if it tolerates some gaming of incentives. The promise of patient values and goals as the driver of patient-centred care is now two decades in development.1 Pragmatic, empirically supported processes for identifying patient goals and preferences during can lasix make you constipated routine care and aligning treatment decisions to achieve these patient priorities are a welcome addition to the literature. Medicare and health insurers must now respond with incentives and quality measures that promote this mature vision of patient-centred care..

Medication errors have been a leading cause of preventable harm generic lasix online for decades. Assiri and colleagues report that the cost of medication error worldwide exceeds $42 billion, or approximately 5%–6% of all hospitalisations.1 While this topic has been closely studied since its first appearance in scientific literature in 1953,2 the problems continue to evolve alongside changes to the medication-use generic lasix online system. The medication-use system is a function of many elements.

Widespread transitions from paper-based to electronic health records have affected drug ordering and prescribing, documentation, transcribing, dispensing, administering and monitoring in ways that challenge traditional approaches to reducing errors that predate electronic records.3 In addition, the introduction of over 7000 branded small molecules or biologics, generics and biosimilars that overlap numerous therapeutic areas increased dependence on specialty care for people with multiple chronic conditions, and navigating transitions throughout the range of primary to generic lasix online quaternary care have all complicated the ability of health systems to manage individual patient medication needs safely.4 Thus, solutions to address common medication errors 10 or 20 years ago may quickly become outdated in our fast-paced healthcare sector.Medication errors can either be intercepted prior to reaching the patient or produce adverse drug events (ADEs) ranging from benign to life-threatening. Concerning prevalence rates of ADEs in hospitalised patients have been reported at 3.22% in the UK, 4.78% in Germany and 5.64% in the USA.5 For a country the size of the USA, the US Food and Drug Administration reports that this rate represents over 100 000 ADEs per year generic lasix online. However, these data relate only to the more severe ADEs.

Those resulting in death, a life threatening health state, hospitalisation, disability or birth defect.6 These figures therefore encapsulate pain and suffering as captured in generic lasix online administrative data but do not include the multitude of patients who missed one or more days of work or school, developed symptoms necessitating an outpatient or emergency room visit, induced long-term harm, or the attendant health system costs. The data therefore give only part of the overall picture.In contrast, based on a comprehensive analysis of UK data, the study by Elliott and colleagues in this issue attempts to illustrate the true full impact of medication errors and the associated risk of ADEs.7 Of the 237 million medication errors estimated to occur in England each year, 66 million are potentially clinically significant and result in 181 thousand hospital days and 1708 deaths at the cost of £98 million to the National Health Service. However, the generic lasix online aetiology and factors influencing medication errors that lead to these ADEs exceed ‘ubiquitous medicine use’ in the country.

That is, generic lasix online the causes of ADEs are multifaceted. In this case, comprehensive improvement of the medication-use system should not be overlooked—and its multifaceted nature is likely to require the execution of quality improvement initiatives across many domains.Elliott and colleagues break down medication errors by stage within the medication-use system to highlight the degree to which these issues are multifaceted. It comes as little surprise that across primary care, secondary care and care homes, prescribing, dispensing, administration and monitoring generic lasix online errors are prominent.

However, the degree to which data are missing is also concerning and therefore may underestimate the prevalence and costs of medication errors. How can any health system, let alone an entire generic lasix online National Health Service devise best practices to reduce medication errors when data that present a substantial proportion of variability in ADEs are missing?. ‘No UK data available’ in tables throughout Elliott and colleagues’ paper (ie, no comparable UK data were available for particular settings, such as care homes) is as insightful as the numbers that are displayed since it presents an opportunity to improve quality of care informed by an investment in better data, among other needs.As with any quality improvement initiative, beginning with a framework to reduce ADEs as a generic lasix online result of medication error requires an established structure.8 The ‘five rights’ of medication administration offer health systems one potential structure on which to ensure individuals receive the right treatment to maximise clinical benefit and minimise harm.

The right patient, the right drug, the right dose, the right route and the right time.9 Building from these principles, it becomes apparent that methods and technologies for interdiction of medication error and preventable ADEs are still being refined along with variability in execution. Relatively simple solutions such as clear prescription labelling and safe packaging, multiple prescriber and pharmacy tracking to capture drug interaction risk, along with information sharing and advances in drug therapy stewardship, are examples of processes around which to build a quality improvement programme from the five rights structure that may achieve reduced rates generic lasix online of ADEs.4 Further targeting of these improvements within health system components where medication errors are most common, such as ambulatory and primary care settings and transitions of care, would represent efficient use of healthcare resources to reduce ADEs.1By addressing issues in primary care and outpatient settings, the healthcare sector would also minimise the number of ADEs that result in more expensive secondary, tertiary and quaternary care, thereby increasing the probability of additional drug–drug interactions or other risks of medication errors. Further to this are settings and spaces where prescription practices are engaged, fulfilled and monitored.

Providers and pharmacists rarely coexist in the same clinical settings in primary, outpatient and ambulatory care as they do in tertiary and quaternary care where the medical community has already recognised the importance of including pharmacists in patient rounds to review and reconcile medication errors.10 Past studies have noted that when the pharmacist is part of a clinical team to address patient needs within complex medication strategies, reductions in ADEs can be achieved throughout various healthcare settings.11–13 While the physically aligned presence of providers and pharmacists may not be as straightforward to facilitate in primary and outpatient care, increased telecommunication throughout the medication use process, including computer order entry and medication reconciliation, could resolve issues that generic lasix online may otherwise lead to medication errors and subsequent ADEs.As the research of Elliott et al7 and other findings highlight, ADEs are a costly, harmful issue that remains prevalent in global healthcare. The added complexity created by layering healthcare delivery across many settings of primary and specialty care creates gaps in communication where prescribers generic lasix online lack means or availability to actively communicate with pharmacists to identify and resolve potential medication errors. The sheer increase in volumes of prescription medications that outpaces process efficiencies also challenges the ability of these two stakeholders to communicate directly on a per-patient basis.

However, medication reviews focused on patients who take multiple prescriptions, have debilitating long-term conditions or have recently experienced acute decompensation that could make them particularly vulnerable to repeat episodes are an important focus for whom to narrow the degree of communication by default over medication review.14Beyond these suggestions for quality improvement based on current information, the study by Elliott and colleagues highlights the need for additional data to further direct efforts towards efficient means of sustaining generic lasix online reduced ADE rates. Missing data are prevalent throughout the field of ADE outcomes, either because medication errors fail to meet the threshold that institutions such as the US Food and Drug Administration set for a sentinel event or because such errors go completely unnoticed generic lasix online without being recorded as an episode within the health system. Many nations facing the reality of spending millions on ADEs could more proactively invest in improved reporting systems to precisely capture medication errors data, and which instances lead to minor as opposed to major ADEs, and the systems and clinical factors predicting them.

These investments in better and broader data collection and quality improvement programme implementation often frighten away health system generic lasix online directors who fail to recognise the balance between action and reaction. Elliott and colleagues’ expected value of the economic burden of ADE is almost certainly an underestimate. If much of the data generic lasix online on ADEs are missing from the UK system, especially at transitions of care, and other ADEs go under-reported, then the current estimate of £98 million per annum is lower than the true medical and societal cost of this issue, including non-monetary clinical disutility.

The alternative cost scenario that Elliott and colleagues present in the range of £728 million per annum is perhaps a more realistic figure and one that justifies spending on quality improvement programming to offset hundreds of millions in avoidable costs.Thus, reporting systems that captures a wider range of ADEs, coupled with improved modes of communication between providers and pharmacists, as well as a systematic effort to conduct root cause analysis that assist health systems to identify the nature of ADEs and evaluate generic lasix online potential solutions, are possibly cost-effective investments.15 The value of this information is imperative to inform more elaborate systems of medication management and target points of communication between providers and pharmacists to reconcile potential instances of medication error.16 Putting a learning health system model into place such as this—perhaps facilitated by machine learning—makes it more likely that damaging medication errors become more a part of our past history than an issue that the medical literature continues to review.For the past two decades, patient-centredness has served as one of six acknowledged dimensions of healthcare quality.1 Initially, healthcare institutions described patient centredness superficially—clean waiting rooms, hotel-like bed and board, access to innovative medical technology—and measured it with crude satisfaction scales. The concept of patient-centred care evolved into a model attuned to the patient experience of care, defined by the interactions between patients and providers and the care environment.2 This patient experience model of patient-centred care has deep normative roots around principles of the patient as the locus of control and a demand for individualisation and customisation of care based on the patient rather than clinician.3 Empirically, patient experience is associated with health outcomes when defined and measured in a timely manner as a specific care experience or interaction between a patient and a healthcare provider.4 The importance of honouring the patient experience is now a widely appreciated construct and a common measure of healthcare quality with a deep evidence base.5 The Hospital Consumer Assessment of Healthcare Providers and Systems, Consumer Assessment of Health Providers and Systems Survey and Press Ganey patient satisfaction measures are ubiquitous measures of quality defining patient experiences of care.Moving beyond patient experience measuresThe effort to transform healthcare systems from clinician to patient centred is not complete. Honouring, measuring and ameliorating patients’ experiences of care is necessary but not sufficient and represents only the first stop on the journey to patient-centred care.6 The second stop is one that nests the locus of control generic lasix online with patients and caregivers.

Patients’ control over healthcare decisions is useful only when transparency exists in all aspects of care. Evidence, costs, processes, outcomes and errors.3 Unfortunately, claims that patients should have control and transparent understanding of all aspects of care have largely been ignored due to institutional inertia, lack of financial incentives and the primacy generic lasix online of professionals. In essence, there are few incentives to change this orientation, and clinicians too often perceive confrontation and frustration rather than partnership.7The primacy of physician professionalism stems from professional control over scientific knowledge and nurse professionalism from control over the practice environment, both bolstered by generic lasix online years of training and experience.

This professional model held for nearly a century when acute illnesses were the primary reason people sought medical care with the assumption that treatments were focused on cure (return to health) and/or alleviation of symptoms (removal of the disease).8 In contrast, healthcare in the 21st century primarily focuses on managing chronic diseases for which there are few cures. In the context of multiple chronic conditions (multimorbidity), the desired outcomes of healthcare are no longer obvious because they extend generic lasix online beyond the goals of curing diseases or prolonging life. Multimorbidity also produces trade-offs among treatments, conditions and possible outcomes.9 For patients with multimorbidity, evidenced-based treatments are often lacking and, when present, there may be conflicts or incongruences across conditions.10 Effective management of chronic conditions requires active, ongoing participation by patients and caregivers outside of healthcare settings.

The intensity of this management can be burdensome, further impacting patient experiences and even outcomes.11 Healthcare professionals now increasingly understand the need generic lasix online to share the burden of treatment decisions with their multimorbid patients.Patient centredness as healthcare that achieves patient prioritiesThe next stop on the journey to patient-centred care is the establishment of collaborative partnerships between healthcare professionals and patients.6 Productive partnerships require a medium for shared understanding that does not default to professional expertise and clinical practice guidelines. We have asserted that patient priorities are the necessary medium for focusing collaboration, discussions and healthcare decisions, especially in the context of complex, chronic illnesses.10 We precisely define patient priorities as the combination of the specific and realistic outcomes and activities (health outcome goals) that individuals want based on what matters most to them and the healthcare activities, including medications, self-care, tests and visits that they are willing and able to perform (healthcare preferences) to achieve their outcome goals.12 Evidence and professional judgement still guide which treatments are relevant, but clinicians should partner with their patients to select and adjust care based on a health goal as opposed to individual disease states.13 Pragmatic studies demonstrate that this patient priorities approach to care reduces polypharmacy and patient-reported treatment burden while increasing care that aligns with patient goals.14 15 Patients and clinicians describe this process as practical and beneficial.16Measuring goal attainment as a patient-centred care quality measureTo promote and generic lasix online disseminate patient priorities-aligned care, novel quality measures are necessary. These quality metrics would evaluate the process for collaboratively identifying patient goals and care preferences and the degree to which patient goals are attained.

In the current issue of BMJ Quality and Safety, Giovannetti et al17 describe the results of an innovative study that evaluated the generic lasix online feasibility of two different approaches to developing quality measures of goals-based care. The study assessed the implementation of these measures into diverse clinical settings and the subsequent interpretability and usefulness of the measures based on the data generated from either approach.As Giovannetti and colleagues describe, the key gap in evaluating goals-based care is the presence of measures for setting and documenting goals as well as tracking goal progress and attainment.17 In routine care, patient goals and care preferences are infrequently and haphazardly written and communicated, often conflicting, and typically focus on end-of-life care or chronic disease biomarkers.18–21 To address these gaps, the authors adapted goal attainment scaling, a reliable and valid approach for measuring goal setting and goal attainment in research studies.22 23 The authors asked patients and clinicians to jointly set a goal and define a set of possible outcomes along a five-point scale. They later discussed and then individually rated the degree of generic lasix online goal attainment.

The other approach evaluated by Giovannetti generic lasix online and colleagues17 is the use of patient-reported outcome measures (PROMs), which are often used to measure specific domains (eg, mood, functioning, symptoms and so on) of health-related quality of life.24–26 In their study, Giovannetti et al17 asked patients and clinicians to jointly set a goal and then select a PROM that best matches that goal. At follow-up, the patient completed the same PROM to assess change over time. Patients and clinicians were given a generic lasix online dozen PROMs from which to select.The study design and results of the study by Giovannetti et al are both novel and provocative.

The authors found that clinicians were more likely to implement goal attainment scaling, noted to be practical to implement, compared generic lasix online with the PROM approach. Furthermore, clinicians found goal attainment scaling more useful for determining which services and supports to recommend and for helping patients achieve their goals. Contrary to common assumptions, the authors found that clinicians and patients set generic lasix online goals collaboratively and focused on patient-centred outcomes rather than disease processes or biomarkers.

These findings suggest that implementation of a goals-based approach in routine care is feasible and demonstrate promise for fostering the shift from disease to patient-centred care.The lack of appeal for the PROM approach is surprising given their broad acceptance as quality measures.27 PROMs are effective tools for measuring particular behaviours, activities or symptoms that are either specific to a disease, such as diabetes,28 or reflect overall health-related quality of life.29 As quality metrics, PROMs provide patient-centred measures that can be applied across a population of patients, such as the Patient Health Questionnaire for measuring depression symptoms. However, patients and generic lasix online clinicians seem to prefer goals-based approaches, such as goal attainment scaling30 and patient priorities care,10 because they better reflect the goals of specific individuals within the context of their own lives. We have shown that when older patients set goals that are specific to their individual lives, they typically fall into one of four generic lasix online health-related values categories.

(1) social and spiritual connections, (2) functioning and independence, (3) life enjoyment and pleasurable activities and (4) balancing quality and quantity of life (managing health).31 32 We have trained clinicians to identify specific and realistic goals based on what matters most to patients by initiating conversations around the four health values categories.12 These conversations can be efficiently incorporated in clinic visits and during telehealth encounters. In another clinical trial, we demonstrated that a patient goals-based approach can significantly improve scores on a validated depression-specific PROM compared with routine guidelines-based care.33 These findings suggest that individualised approaches to goal attainment can be coupled with PROMs to provide a balanced (individualised generic lasix online goals along with population-level measures) approach to quality measurement of patient-centred care.Financial incentives to promote patient-centred careTo facilitate dissemination of patient priorities aligned care, health insurers should support targeted financial incentives to facilitate widespread adoption into routine care. First, time-based reimbursement for clinical encounters with patients is vital.

Medicare’s care management billing codes for annual wellness, advanced care planning and chronic care management are also potential options generic lasix online. Establishment of novel value-based care management codes that are specific to priorities setting and measuring goal generic lasix online progress and attainment would be key drivers of this effort. Furthermore, these codes should support involvement of a range of health professionals.

Training opportunities supported by continuing education credits would generic lasix online further promote patient priorities care. Common concerns about quality measures focused on goal attainment include the setting of unrealistic or inappropriate goals, playing the system with easily attained goals and the nuances of patient–caregiver–clinician goal alignment. These are all practical challenges to achieving a mature generic lasix online goals-aligned care process.

However, at this early stage of development, Medicare generic lasix online should promote all efforts to implement value-based care management codes even if they are used primarily for financial incentives. Any impetus that encourages goal-based conversations and goal setting among patients, caregivers and clinicians will promote the necessary paradigm shift from guidelines-based care to goals-based care even if it tolerates some gaming of incentives. The promise of patient values and goals as the driver of patient-centred care is now two decades in development.1 Pragmatic, empirically supported generic lasix online processes for identifying patient goals and preferences during routine care and aligning treatment decisions to achieve these patient priorities are a welcome addition to the literature.

Medicare and health insurers must now respond with incentives and quality measures that promote this mature vision of patient-centred care..

Lasix overdose

Wealthy nations must do much more, much faster.The United Nations General Assembly lasix overdose in September 2021 will bring countries together at a critical time for marshalling collective action to tackle the global environmental crisis. They will meet again at the biodiversity summit in Kunming, China, and the climate conference (Conference of the lasix overdose Parties (COP)26) in Glasgow, UK. Ahead of these pivotal meetings, we—the editors of health journals worldwide—call for urgent action to keep average global temperature increases below 1.5°C, halt the destruction of nature and protect health.Health is already being harmed by global temperature increases and the destruction of the natural world, a state of affairs health professionals have been bringing attention to for decades.1 The science is unequivocal. A global increase of 1.5°C above the preindustrial average and the continued loss of biodiversity risk catastrophic harm to health that will be impossible to reverse.2 3 Despite lasix overdose the world’s necessary preoccupation with hypertension medications, we cannot wait for the lasix to pass to rapidly reduce emissions.Reflecting the severity of the moment, this editorial appears in health journals across the world. We are united in recognising that only fundamental and equitable changes to societies will reverse our current lasix overdose trajectory.The risks to health of increases above 1.5°C are now well established.2 Indeed, no temperature rise is ‘safe’.

In the past 20 years, heat-related mortality among people aged over 65 has increased by more than 50%.4 Higher temperatures have brought increased dehydration and renal function loss, dermatological malignancies, tropical s, adverse mental health outcomes, pregnancy complications, allergies, and cardiovascular and pulmonary morbidity and mortality.5 6 Harms disproportionately affect the most vulnerable, including children, older populations, ethnic minorities, poorer communities and those with underlying health problems.2 4Global heating is also contributing to the decline in global yield potential for major crops, falling by 1.8%–5.6% since 1981. This, together with the effects of extreme weather and soil depletion, is hampering efforts to reduce undernutrition.4 Thriving ecosystems are essential to human lasix overdose health, and the widespread destruction of nature, including habitats and species, is eroding water and food security and increasing the chance of lasixs.3 7 8The consequences of the environmental crisis fall disproportionately on those countries and communities that have contributed least to the problem and are least able to mitigate the harms. Yet no country, no matter how wealthy, can shield itself from these impacts. Allowing the consequences to fall disproportionately on the most vulnerable will breed more conflict, food insecurity, forced displacement and zoonotic disease, with severe implications for lasix overdose all countries and communities. As with the hypertension medications lasix, we are globally as strong as our weakest member.Rises above 1.5°C increase the chance of reaching tipping points in natural systems that could lock the world into an acutely unstable lasix overdose state.

This would critically impair our ability to mitigate harms and to prevent catastrophic, runaway environmental change.9 10Global targets are not enoughEncouragingly, many governments, financial institutions and businesses are setting targets to reach net-zero emissions, including targets for 2030. The cost of renewable energy lasix overdose is dropping rapidly. Many countries are aiming to protect at least 30% of the world’s land and oceans by 2030.11These promises are not enough lasix overdose. Targets are easy to set and hard to achieve. They are yet to be matched with credible short-term and longer-term plans to accelerate lasix overdose cleaner technologies and transform societies.

Emissions reduction plans do not adequately incorporate health considerations.12 Concern is growing that temperature rises above 1.5°C are beginning to be seen as inevitable, or even acceptable, to powerful members of the global community.13 Relatedly, current strategies for reducing emissions to net zero by the middle of the century implausibly assume that the world will acquire great capabilities to remove greenhouse gases from the atmosphere.14 15This insufficient action means that temperature increases are likely to be well in excess of 2°C,16 a catastrophic outcome for health and environmental stability. Critically, the destruction of nature does not have parity of esteem with the climate element of the crisis, and every single global target to restore biodiversity loss by 2020 was missed.17 This is an overall environmental crisis.18Health professionals are united with environmental scientists, businesses lasix overdose and many others in rejecting that this outcome is inevitable. More can and must be done now—in Glasgow and Kunming—and in the immediate years lasix overdose that follow. We join health professionals worldwide who have already supported calls for rapid action.1 19Equity must be at the centre of the global response. Contributing a fair share to the global effort means that reduction commitments must account for the cumulative, historical contribution lasix overdose each country has made to emissions, as well as its current emissions and capacity to respond.

Wealthier countries will have to cut emissions more quickly, making reductions by 2030 beyond those currently proposed20 21 and reaching net-zero emissions before 2050. Similar targets and emergency action are needed for biodiversity loss and the wider destruction of the natural world.To achieve these targets, governments lasix overdose must make fundamental changes to how our societies and economies are organised and how we live. The current strategy of encouraging lasix overdose markets to swap dirty for cleaner technologies is not enough. Governments must intervene to support the redesign of transport systems, cities, production and distribution of food, markets for financial investments, health systems, and much more. Global coordination is needed to ensure that the rush for cleaner technologies does not come at the cost of lasix overdose more environmental destruction and human exploitation.Many governments met the threat of the hypertension medications lasix with unprecedented funding.

The environmental crisis demands a similar lasix overdose emergency response. Huge investment will be needed, beyond what is being considered or delivered anywhere in the world. But such investments will produce huge positive health and economic outcomes lasix overdose. These include high-quality jobs, reduced air pollution, increased physical activity, and improved housing and diet. Better air quality alone would realise health benefits that easily offset the global costs of emissions reductions.22These measures will also improve the social and economic determinants of health, the poor state of which may have made populations more vulnerable to the hypertension medications lasix.23 But the changes cannot be achieved through a return to damaging austerity policies or the continuation of the large inequalities of wealth and power within and between lasix overdose countries.Cooperation hinges on wealthy nations doing moreIn particular, countries that have disproportionately created the environmental crisis must do more to support low-income and middle-income countries to build cleaner, healthier and more resilient societies.

High-income countries must meet and go beyond their outstanding commitment to provide $100 billion a year, making up for any shortfall lasix overdose in 2020 and increasing contributions to and beyond 2025. Funding must be equally split between mitigation and adaptation, including improving the resilience of health systems.Financing should be through grants rather than loans, building local capabilities and truly empowering communities, and should come alongside forgiving large debts, which constrain the agency of so many low-income countries. Additional funding must be lasix overdose marshalled to compensate for inevitable loss and damage caused by the consequences of the environmental crisis.As health professionals, we must do all we can to aid the transition to a sustainable, fairer, resilient and healthier world. Alongside acting to reduce the harm from the environmental crisis, we should proactively contribute to global prevention of further damage and action on the root causes of the crisis. We must hold global leaders to account and continue lasix overdose to educate others about the health risks of the crisis.

We must join in the work to achieve environmentally sustainable health systems before 2040, recognising that this will mean changing clinical lasix overdose practice. Health institutions have already divested more than $42 billion of assets from fossil fuels. Others should join them.4The greatest threat to global public health is the continued lasix overdose failure of world leaders to keep the global temperature rise below 1.5°C and to restore nature. Urgent, society-wide changes must be made and will lead to lasix overdose a fairer and healthier world. We, as editors of health journals, call for governments and other leaders to act, marking 2021 as the year that the world finally changes course.Ethics statementsPatient consent for publicationNot required.AbstractPhenome-wide association study (PheWAS) has been increasingly used to identify novel genetic associations across a wide spectrum of phenotypes.

This systematic review aims to summarise the lasix overdose PheWAS methodology, discuss the advantages and challenges of PheWAS, and provide potential implications for future PheWAS studies. Medical Literature Analysis and Retrieval System Online (MEDLINE) and Excerpta Medica Database (EMBASE) databases were searched to identify all published PheWAS studies up until 24 April 2021. The PheWAS methodology incorporating how lasix overdose to perform PheWAS analysis and which software/tool could be used, were summarised based on the extracted information. A total lasix overdose of 1035 studies were identified and 195 eligible articles were finally included. Among them, 137 (77.0%) contained 10 000 or more study participants, 164 (92.1%) defined the phenome based on electronic medical records data, 140 (78.7%) used genetic variants as predictors, and 73 (41.0%) conducted replication analysis to validate PheWAS findings and almost all of them (94.5%) received consistent results.

The methodology applied in these PheWAS studies was dissected into several lasix overdose critical steps, including quality control of the phenome, selecting predictors, phenotyping, statistical analysis, interpretation and visualisation of PheWAS results, and the workflow for performing a PheWAS was established with detailed instructions on each step. This study provides a comprehensive overview of PheWAS methodology to help practitioners achieve a better understanding of the PheWAS design, to detect understudied or overstudied outcomes, and to direct their research by applying the most appropriate software and online tools for their study data structure.genetic association studiesmolecular epidemiologypublic health.

Wealthy nations must do much more, much faster.The United Nations General Assembly in September generic lasix online 2021 will bring countries visit the website together at a critical time for marshalling collective action to tackle the global environmental crisis. They will meet again at the biodiversity summit generic lasix online in Kunming, China, and the climate conference (Conference of the Parties (COP)26) in Glasgow, UK. Ahead of these pivotal meetings, we—the editors of health journals worldwide—call for urgent action to keep average global temperature increases below 1.5°C, halt the destruction of nature and protect health.Health is already being harmed by global temperature increases and the destruction of the natural world, a state of affairs health professionals have been bringing attention to for decades.1 The science is unequivocal. A global increase of 1.5°C above the preindustrial average and the continued loss of biodiversity risk catastrophic harm to health that will be impossible to reverse.2 3 Despite the world’s necessary preoccupation with hypertension medications, we cannot wait for the lasix to pass to rapidly reduce emissions.Reflecting the severity of the moment, this editorial appears in health journals across the generic lasix online world.

We are united in recognising that only fundamental generic lasix online and equitable changes to societies will reverse our current trajectory.The risks to health of increases above 1.5°C are now well established.2 Indeed, no temperature rise is ‘safe’. In the past 20 years, heat-related mortality among people aged over 65 has increased by more than 50%.4 Higher temperatures have brought increased dehydration and renal function loss, dermatological malignancies, tropical s, adverse mental health outcomes, pregnancy complications, allergies, and cardiovascular and pulmonary morbidity and mortality.5 6 Harms disproportionately affect the most vulnerable, including children, older populations, ethnic minorities, poorer communities and those with underlying health problems.2 4Global heating is also contributing to the decline in global yield potential for major crops, falling by 1.8%–5.6% since 1981. This, together generic lasix online with the effects of extreme weather and soil depletion, is hampering efforts to reduce undernutrition.4 Thriving ecosystems are essential to human health, and the widespread destruction of nature, including habitats and species, is eroding water and food security and increasing the chance of lasixs.3 7 8The consequences of the environmental crisis fall disproportionately on those countries and communities that have contributed least to the problem and are least able to mitigate the harms. Yet no country, no matter how wealthy, can shield itself from these impacts.

Allowing the consequences to fall disproportionately on the most vulnerable will breed more conflict, food insecurity, forced displacement and zoonotic disease, with severe implications for all countries and communities generic lasix online. As with the hypertension medications lasix, we are globally as strong as our weakest member.Rises above generic lasix online 1.5°C increase the chance of reaching tipping points in natural systems that could lock the world into an acutely unstable state. This would critically impair our ability to mitigate harms and to prevent catastrophic, runaway environmental change.9 10Global targets are not enoughEncouragingly, many governments, financial institutions and businesses are setting targets to reach net-zero emissions, including targets for 2030. The cost generic lasix online of renewable energy is dropping rapidly.

Many countries are aiming to protect at least 30% of the generic lasix online world’s land and oceans by 2030.11These promises are not enough. Targets are easy to set and hard to achieve. They are yet to be matched with credible short-term and longer-term plans to accelerate cleaner technologies and generic lasix online transform societies. Emissions reduction plans do not adequately incorporate health considerations.12 Concern is growing that temperature rises above 1.5°C are beginning to be seen as inevitable, or even acceptable, to powerful members of the global community.13 Relatedly, current strategies for reducing emissions to net zero by the middle of the century implausibly assume that the world will acquire great capabilities to remove greenhouse gases from the atmosphere.14 15This insufficient action means that temperature increases are likely to be well in excess of 2°C,16 a catastrophic outcome for health and environmental stability.

Critically, the destruction of nature does not have parity of esteem with the climate element of the crisis, and every single global target to restore biodiversity loss by generic lasix online 2020 was missed.17 This is an overall environmental crisis.18Health professionals are united with environmental scientists, businesses and many others in rejecting that this outcome is inevitable. More can and must be generic lasix online done now—in Glasgow and Kunming—and in the immediate years that follow. We join health professionals worldwide who have already supported calls for rapid action.1 19Equity must be at the centre of the global response. Contributing a fair share to the generic lasix online global effort means that reduction commitments must account for the cumulative, historical contribution each country has made to emissions, as well as its current emissions and capacity to respond.

Wealthier countries will have to cut emissions more quickly, making reductions by 2030 beyond those currently proposed20 21 and reaching net-zero emissions before 2050. Similar targets and emergency action are needed for biodiversity loss and the wider destruction of the natural world.To achieve these targets, governments must make fundamental changes generic lasix online to how our societies and economies are organised and how we live. The current strategy of encouraging markets to swap dirty for cleaner technologies is not enough generic lasix online. Governments must intervene to support the redesign of transport systems, cities, production and distribution of food, markets for financial investments, health systems, and much more.

Global coordination is needed generic lasix online to ensure that the rush for cleaner technologies does not come at the cost of more environmental destruction and human exploitation.Many governments met the threat of the hypertension medications lasix with unprecedented funding. The environmental crisis demands a generic lasix online similar emergency response. Huge investment will be needed, beyond what is being considered or delivered anywhere in the world. But such investments will produce huge positive generic lasix online health and economic outcomes.

These include high-quality jobs, reduced air pollution, increased physical activity, and improved housing and diet. Better air quality alone would realise health benefits that easily offset the global costs of emissions reductions.22These measures will also improve the social and economic determinants of health, the poor state of which may have made populations more vulnerable to the hypertension medications lasix.23 But generic lasix online the changes cannot be achieved through a return to damaging austerity policies or the continuation of the large inequalities of wealth and power within and between countries.Cooperation hinges on wealthy nations doing moreIn particular, countries that have disproportionately created the environmental crisis must do more to support low-income and middle-income countries to build cleaner, healthier and more resilient societies. High-income countries must meet and go beyond their outstanding commitment to provide $100 billion a year, making up for any shortfall in 2020 and increasing generic lasix online contributions to and beyond 2025. Funding must be equally split between mitigation and adaptation, including improving the resilience of health systems.Financing should be through grants rather than loans, building local capabilities and truly empowering communities, and should come alongside forgiving large debts, which constrain the agency of so many low-income countries.

Additional funding must be marshalled to compensate for inevitable loss and damage caused by the consequences of the environmental crisis.As health professionals, we must do all we can generic lasix online to aid the transition to a sustainable, fairer, resilient and healthier world. Alongside acting to reduce the harm from the environmental crisis, we should proactively contribute to global prevention of further damage and action on the root causes of the crisis. We must hold global leaders to account and generic lasix online continue to educate others about the health risks of the crisis. We must generic lasix online join in the work to achieve environmentally sustainable health systems before 2040, recognising that this will mean changing clinical practice.

Health institutions have already divested more than $42 billion of assets from fossil fuels. Others should join them.4The greatest threat generic lasix online to global public health is the continued failure of world leaders to keep the global temperature rise below 1.5°C and to restore nature. Urgent, society-wide changes must be made and will lead to a fairer and healthier generic lasix online world. We, as editors of health journals, call for governments and other leaders to act, marking 2021 as the year that the world finally changes course.Ethics statementsPatient consent for publicationNot required.AbstractPhenome-wide association study (PheWAS) has been increasingly used to identify novel genetic associations across a wide spectrum of phenotypes.

This systematic generic lasix online review aims to summarise the PheWAS methodology, discuss the advantages and challenges of PheWAS, and provide potential implications for future PheWAS studies. Medical Literature Analysis and Retrieval System Online (MEDLINE) and Excerpta Medica Database (EMBASE) databases were searched to identify all published PheWAS studies up until 24 April 2021. The PheWAS methodology incorporating generic lasix online how to perform PheWAS analysis and which software/tool could be used, were summarised based on the extracted information. A total generic lasix online of 1035 studies were identified and 195 eligible articles were finally included.

Among them, 137 (77.0%) contained 10 000 or more study participants, 164 (92.1%) defined the phenome based on electronic medical records data, 140 (78.7%) used genetic variants as predictors, and 73 (41.0%) conducted replication analysis to validate PheWAS findings and almost all of them (94.5%) received consistent results. The methodology applied in these PheWAS studies was dissected into several critical steps, including quality control of the phenome, selecting generic lasix online predictors, phenotyping, statistical analysis, interpretation and visualisation of PheWAS results, and the workflow for performing a PheWAS was established with detailed instructions on each step. This study provides a comprehensive overview of PheWAS methodology to help practitioners achieve a better understanding of the PheWAS design, to detect understudied or overstudied outcomes, and to direct their research by applying the most appropriate software and online tools for their study data structure.genetic association studiesmolecular epidemiologypublic health.

Lasix and erectile dysfunction

Shutterstock Cipro online canada U.S lasix and erectile dysfunction. Rep Joe Morelle (D-NY) announced Tuesday that provisions he authored to help veterans in crisis and reduce veteran suicide rates have been included in the Commander John Scott Hannon Veterans Mental Health Care Improvement Act, signed into law Oct. 17.

€œSuicide claims the lives of over 7,000 veterans every single year – or 20 veterans every day,” Morelle said. €œThis is a crisis that is tearing apart families across America. These brave men and women put everything on the line for our country, and we must ensure they have the tools they need to get the help they deserve.”Sponsored by Sen.

Jon Tester (D-MT), the Hannon Act ensures veterans have access to mental health care resources. The legislation includes provisions of Morelle’s Reach Every Veteran in Crisis Act that was intended to combat veteran suicide. Provisions from Morelle’s bill included in the Hannon Act include.

Ensuring resources already allocated by Congress are used effectively and efficiently. Establishing targets to evaluate the efficacy of mental health and suicide prevention outreach campaigns. Submitting reports on the expenditures and obligations of Veterans’ Health Administration’s Office of Mental Health and Suicide Prevention to the House and Senate Veterans Affairs, and Appropriations Committees.

Morelle wrote the legislation in 2019 after learning the VA had spent a fraction of its suicide prevention media outreach budget and that the agency never established ways to evaluate the efficacy of its suicide prevention campaign. €œDAV is pleased that provisions from Representative Morelle’s legislation The Reach Every Veteran in Crisis Act were included in the recently-passed Commander John Scott Hannon Veterans Mental Health Care Improvement Act of 2019,” said Joy J Ilem, National Legislative Director of Disabled American Veterans. €œEffective mental health and suicide prevention media outreach campaigns are an essential part of combating the crisis of veteran suicide.

We thank Representative Morelle for his efforts to prevent veteran suicide and improve the lives of our nation’s veterans through his legislation aimed at improved oversight and evaluation of VA’s suicide prevention media outreach campaigns.”Shutterstock Arizona recently launched a plan to address suicide rates in the state, an effort led by Dr. Cara Christ, Department of Health Services (ADHS) director and Department of Economic Security interim director.Three main goals encompass the Suicide Prevention Action Plan. The state aims to collect data on suicide to identify the demographic groups most at risk, improve mental health, and ensure treatment and support services are available.The state will work with insurance companies to ensure they cover mental health.

€œIt is heartbreaking for families, friends, and communities when someone dies from suicide and can have a lifelong impact,” Christ said. €œThis action plan calls for a whole community approach to prevent suicide. ADHS will continue to implement statewide strategies that decrease factors that put people at risk of suicide, increase protective factors that promote resilience and coping and ensure those who need help can access it.”ADHS met last year with stakeholders to identify and develop strategies to create a supportive environment and engage communities.Stakeholders included families and individuals impacted by suicide, healthcare providers, mental health professionals, schools, first responders, community partners, government agencies, and tribal nations.

Suicide rates in Arizona increased from 1,304 suicides in 2017 to 1,432 in 2018.Shutterstock Wisconsin collected 89,982 pounds of disposed prescription medications, the largest nationwide, on Drug Take Back Day held Oct. 24, according to Attorney General Josh Kaul.The drugs were collected in 485 permanent drug disposal boxes, and 230 law enforcement collected drugs during Drug Take Back events. The collected drugs were sent to Covanta Energy Corp.

In Indianapolis for incineration.“Thank you to the many Wisconsinites who safely disposed of unused and unwanted medications, making Wisconsin’s Drug Take Back the most successful in the nation,” Kaul said. €œYour efforts help with the fight against substance-use disorder by ensuring that those unused medications won’t be diverted.” Trace amounts of pharmaceuticals have been detected in rivers and lakes, and experts warn the public to never dispose of unused or expired medicine by flushing it down the drain or toilet. Nationwide, Drug Take Back Day is sponsored by the U.S.

Drug Enforcement Administration.Wisconsin’s Drug Take Back Day was possible with the support of numerous organization, including local law enforcement agencies, the Wisconsin State Patrol, the Wisconsin Department of Health Services, the Wisconsin Department of Agriculture, the Waukesha County Sheriff’s Office, Waukesha County, the Wisconsin Department of Natural Resources, the Wisconsin Department of Military Affairs, and the Indiana State Police.Shutterstock Illinois recently became the latest state to join the Governor’s Challenge to Prevent Suicide, a challenge that champions mental health support and preventative services for veterans.The Illinois Department of Veterans Affairs (IDVA) and the federal Substance Abuse and Mental Health Services Administration will dedicate $2 million to the initiative.The state will use the latest public health research and data to create and implement best practices. It will also target those most impacted by mental illness, such as veterans and communities of color.In Illinois, suicide is the third leading cause of death for residents less than 25 years old and the 12th leading cause of death for all age groups.“We must do everything within our power as policymakers to fight for the lives of our nation’s heroes just as they have fought for us,” Linda Chapa LaVia, IDVA director, said. €œI applaud Gov.

(JB) Pritzker for rising to the challenge to work towards stamping out veteran suicide once and for all. Veterans and active-duty service members are at an even more increased risk of taking their own lives during this isolating time in our state’s history. This funding to put concrete plans into action could not come at a better time.” There are 27 states participating in the challenge.Shutterstock Calling the terms of a settlement between the U.S.

Department of Justice and Purdue Pharma “inappropriate use of federal authority,” four Democratic Senators Tuesday urged U.S. Attorney General Bill Barr to reconsider the agreement. U.S.

Sens. Tammy Baldwin (D-WI), Sheldon Whitehouse (D-RI), Maggie Hassan (D-NH), and Elizabeth Warren (D-MA) took issue with a provision in the $8 billion settlement that would turn Purdue’s business into a public benefit company after it emerges from bankruptcy. The senators said they, along with 25 attorneys general from across the country, object to the idea that local and state governments would-be owners of a company that continues to produce a drug that has killed thousands of their constituents.

€œWe write to raise concerns about a key element of the Department of Justice’s (DOJ) settlement agreement with Purdue Pharma (Purdue) announced on Wednesday, October 21, 2020. We ask that you defer court approval of the proposed agreement until the appropriate stakeholders have addressed public policy concerns associated with the agreement, which all but requires Purdue to emerge from bankruptcy as a public benefit company (PBC), to function “entirely in the public interest,” with proceeds directed toward State and local governments. This arrangement ignores the objections of many of the States themselves, who have no interest in owning or operating a company that has devastated their communities with dangerous opioids, and raises significant public policy concerns,” the Senators in their letter.

The senators said the idea, generated by Purdue and the Sackler family, would give the company protection. €œPurdue and the Sackler family are the driving force behind the inclusion of the PBC in the agreement and had proposed that it be included during the company’s ongoing bankruptcy proceedings. Allowing Purdue to emerge from bankruptcy as a PBC would enable it to shed its liability while continuing to manufacture and sell opioids, with its creditors—including state and local governments who have sued Purdue for the harms it caused—owning a stake in its profits,” the senators wrote.

The senators also questioned the efficacy of the deal, writing that the agreement would allow Purdue to give states less money upfront and promise to pay the remaining terms of the settlement out of future profits that are uncertain and may never cover the full value of the settlement. €œStates would get less money immediately and, because these profits are uncertain, may never recover the full value of the settlement. At a minimum, the PBC also creates the appearance of a conflict of interest, as citizens may wonder whether their government will effectively regulate a company in which it has a financial interest.

In a worst case, aligning the financial interests of States with the increasing sale of opioids, which is the very reason the lawsuit was brought against Purdue in the first place, could significantly and negatively impact public health,” they wrote. The senators called on Barr to defer court approval of the settlement until states and local governments and other stakeholders have addressed the issues..

Shutterstock http://glasswing.org/cipro-online-canada/ generic lasix online U.S. Rep Joe Morelle (D-NY) announced Tuesday that provisions he authored to help veterans in crisis and reduce veteran suicide rates have been included in the Commander John Scott Hannon Veterans Mental Health Care Improvement Act, signed into law Oct. 17.

€œSuicide claims the lives of over 7,000 veterans every single year – or 20 veterans every day,” Morelle said. €œThis is a crisis that is tearing apart families across America. These brave men and women put everything on the line for our country, and we must ensure they have the tools they need to get the help they deserve.”Sponsored by Sen.

Jon Tester (D-MT), the Hannon Act ensures veterans have access to mental health care resources. The legislation includes provisions of Morelle’s Reach Every Veteran in Crisis Act that was intended to combat veteran suicide. Provisions from Morelle’s bill included in the Hannon Act include.

Ensuring resources already allocated by Congress are used effectively and efficiently. Establishing targets to evaluate the efficacy of mental health and suicide prevention outreach campaigns. Submitting reports on the expenditures and obligations of Veterans’ Health Administration’s Office of Mental Health and Suicide Prevention to the House and Senate Veterans Affairs, and Appropriations Committees.

Morelle wrote the legislation in 2019 after learning the VA had spent a fraction of its suicide prevention media outreach budget and that the agency never established ways to evaluate the efficacy of its suicide prevention campaign. €œDAV is pleased that provisions from Representative Morelle’s legislation The Reach Every Veteran in Crisis Act were included in the recently-passed Commander John Scott Hannon Veterans Mental Health Care Improvement Act of 2019,” said Joy J Ilem, National Legislative Director of Disabled American Veterans. €œEffective mental health and suicide prevention media outreach campaigns are an essential part of combating the crisis of veteran suicide.

We thank Representative Morelle for his efforts to prevent veteran suicide and improve the lives of our nation’s veterans through his legislation aimed at improved oversight and evaluation of VA’s suicide prevention media outreach campaigns.”Shutterstock Arizona recently launched a plan to address suicide rates in the state, an effort led by Dr. Cara Christ, Department of Health Services (ADHS) director and Department of Economic Security interim director.Three main goals encompass the Suicide Prevention Action Plan. The state aims to collect data on suicide to identify the demographic groups most at risk, improve mental health, and ensure treatment and support services are available.The state will work with insurance companies to ensure they cover mental health.

€œIt is heartbreaking for families, friends, and communities when someone dies from suicide and can have a lifelong impact,” Christ said. €œThis action plan calls for a whole community approach to prevent suicide. ADHS will continue to implement statewide strategies that decrease factors that put people at risk of suicide, increase protective factors that promote resilience and coping and ensure those who need help can access it.”ADHS met last year with stakeholders to identify and develop strategies to create a supportive environment and engage communities.Stakeholders included families and individuals impacted by suicide, healthcare providers, mental health professionals, schools, first responders, community partners, government agencies, and tribal nations.

Suicide rates in Arizona increased from 1,304 suicides in 2017 to 1,432 in 2018.Shutterstock Wisconsin collected 89,982 pounds of disposed prescription medications, the largest nationwide, on Drug Take Back Day held Oct. 24, according to Attorney General Josh Kaul.The drugs were collected in 485 permanent drug disposal boxes, and 230 law enforcement collected drugs during Drug Take Back events. The collected drugs were sent to Covanta Energy Corp.

In Indianapolis for incineration.“Thank you to the many Wisconsinites who safely disposed of unused and unwanted medications, making Wisconsin’s Drug Take Back the most successful in the nation,” Kaul said. €œYour efforts help with the fight against substance-use disorder by ensuring that those unused medications won’t be diverted.” Trace amounts of pharmaceuticals have been detected in rivers and lakes, and experts warn the public to never dispose of unused or expired medicine by flushing it down the drain or toilet. Nationwide, Drug Take Back Day is sponsored by the U.S.

Drug Enforcement Administration.Wisconsin’s Drug Take Back Day was possible with the support of numerous organization, including local law enforcement agencies, the Wisconsin State Patrol, the Wisconsin Department of Health Services, the Wisconsin Department of Agriculture, the Waukesha County Sheriff’s Office, Waukesha County, the Wisconsin Department of Natural Resources, the Wisconsin Department of Military Affairs, and the Indiana State Police.Shutterstock Illinois recently became the latest state to join the Governor’s Challenge to Prevent Suicide, a challenge that champions mental health support and preventative services for veterans.The Illinois Department of Veterans Affairs (IDVA) and the federal Substance Abuse and Mental Health Services Administration will dedicate $2 million to the initiative.The state will use the latest public health research and data to create and implement best practices. It will also target those most impacted by mental illness, such as veterans and communities of color.In Illinois, suicide is the third leading cause of death for residents less than 25 years old and the 12th leading cause of death for all age groups.“We must do everything within our power as policymakers to fight for the lives of our nation’s heroes just as they have fought for us,” Linda Chapa LaVia, IDVA director, said. €œI applaud Gov.

(JB) Pritzker for rising to the challenge to work towards stamping out veteran suicide once and for all. Veterans and active-duty service members are at an even more increased risk of taking their own lives during this isolating time in our state’s history. This funding to put concrete plans into action could not come at a better time.” There are 27 states participating in the challenge.Shutterstock Calling the terms of a settlement between the U.S.

Department of Justice and Purdue Pharma “inappropriate use of federal authority,” four Democratic Senators Tuesday urged U.S. Attorney General Bill Barr to reconsider the agreement. U.S.

Sens. Tammy Baldwin (D-WI), Sheldon Whitehouse (D-RI), Maggie Hassan (D-NH), and Elizabeth Warren (D-MA) took issue with a provision in the $8 billion settlement that would turn Purdue’s business into a public benefit company after it emerges from bankruptcy. The senators said they, along with 25 attorneys general from across the country, object to the idea that local and state governments would-be owners of a company that continues to produce a drug that has killed thousands of their constituents.

€œWe write to raise concerns about a key element of the Department of Justice’s (DOJ) settlement agreement with Purdue Pharma (Purdue) announced on Wednesday, October 21, 2020. We ask that you defer court approval of the proposed agreement until the appropriate stakeholders have addressed public policy concerns associated with the agreement, which all but requires Purdue to emerge from bankruptcy as a public benefit company (PBC), to function “entirely in the public interest,” with proceeds directed toward State and local governments. This arrangement ignores the objections of many of the States themselves, who have no interest in owning or operating a company that has devastated their communities with dangerous opioids, and raises significant public policy concerns,” the Senators in their letter.

The senators said the idea, generated by Purdue and the Sackler family, would give the company protection. €œPurdue and the Sackler family are the driving force behind the inclusion of the PBC in the agreement and had proposed that it be included during the company’s ongoing bankruptcy proceedings. Allowing Purdue to emerge from bankruptcy as a PBC would enable it to shed its liability while continuing to manufacture and sell opioids, with its creditors—including state and local governments who have sued Purdue for the harms it caused—owning a stake in its profits,” the senators wrote.

The senators also questioned the efficacy of the deal, writing that the agreement would allow Purdue to give states less money upfront and promise to pay the remaining terms of the settlement out of future profits that are uncertain and may never cover the full value of the settlement. €œStates would get less money immediately and, because these profits are uncertain, may never recover the full value of the settlement. At a minimum, the PBC also creates the appearance of a conflict of interest, as citizens may wonder whether their government will effectively regulate a company in which it has a financial interest.

In a worst case, aligning the financial interests of States with the increasing sale of opioids, which is the very reason the lawsuit was brought against Purdue in the first place, could significantly and negatively impact public health,” they wrote. The senators called on Barr to defer court approval of the settlement until states and local governments and other stakeholders have addressed the issues..