Tab lasix 40mg price in canada
Activating mutations in receptor guanylyl cyclase C (GC-C), the target of gastrointestinal peptide hormones guanylin and uroguanylin, and bacterial tab lasix 40mg price in canada heat-stable enterotoxins 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 mouse model tab lasix 40mg price in canada 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, tab lasix 40mg price in canada 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 tab lasix 40mg price in canada to pediatric 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 tab lasix 40mg price in canada genes associated with very earlyâonset IBD are mutations in the receptor guanylyl cyclase C gene (GUCY2C.
Bose et al., 2020. Crowley et al., 2020). The receptor encoded by this gene, guanylyl cyclase C (GC-C), is tab lasix 40mg price in canada the target of the gastrointestinal hormones guanylin (encoded by GUCA2A) and uroguanylin (encoded by GUCA2B. Arshad and Visweswariah, 2012. Basu et tab lasix 40mg price in canada 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 3â²5â²-cyclic guanosine monophosphate (cGMP) levels in the intestinal cell and activation of cGMP-dependent protein kinase II (PKGII. Lohmann et al., 1997) tab lasix 40mg price in canada. PKGII phosphorylates the cystic fibrosis transmembrane conductance regulator (CFTR) and the sodium-hydrogen exchanger, NHE3 (Chen et al., 2015. Golin-Bisello et tab lasix 40mg price in canada al., 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 tab lasix 40mg price in canada from the cell required for 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 tab lasix 40mg price in canada resulted in 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 tab lasix 40mg price in canada 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 tab lasix 40mg price in canada al., 2018).
Patients suffering from FGDS and children with de novo mutations in GUCY2C present with Crohnâs disease (CD)âlike symptoms and colitis in addition to diarrhea (Fiskerstrand et al., 2012. Müller et tab lasix 40mg price in canada 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 Administrationâapproved 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 tab lasix 40mg price in canada 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 apparent signs of constipation, reports indicate that they demonstrate neurological tab lasix 40mg price in canada 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-Câ/â mice are more susceptible to oral Salmonella enterica enterica serovar Typhimurium (Majumdar et al., 2018). Inactivating mutations have been reported in GUCY2C, with tab lasix 40mg price in canada 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 tab lasix 40mg price in canada 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 watery feces, regulation of Cftr and Nhe3 tab lasix 40mg price in canada 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 secretory diarrhea associated with IBD and the more prevalent infectious diarrheal illness caused by tab lasix 40mg price in canada 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 tab lasix 40mg price in canada of synthesis of guanylin and uroguanylin.
Therefore, serum levels of the propeptides are reflective of levels produced in the gut. However, we tab lasix 40mg price in canada did not see 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 cells tab lasix 40mg price in canada (Fig.
2 C). The presence of GC-C harboring hyperactivating mutations in patientsâ gut was hypothesized to result in elevated intra-epithelial cell cGMP in response tab lasix 40mg price in canada 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 tab lasix 40mg price in canada â¼5- 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 tab lasix 40mg price in canada a delayed gut transit time due to regurgitation of gut contents in the small intestine (von Volkmann et al., 2017, 2016). We estimated gut transit in wild type and S839I mice but observed no change in S839I mice (Fig. 2 E) tab lasix 40mg price in canada. 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) tab lasix 40mg price in canada. The water content in feces produced by S839I mice was higher (Fig. 2 G) tab lasix 40mg price in canada. 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 tab lasix 40mg price in canada. Golin-Bisello et al., 2005. Lin et al., 1992) tab lasix 40mg price in canada. 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 tab lasix 40mg price in canada that some effects of cGMP in the colon could be 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) tab lasix 40mg price in canada. 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 tab lasix 40mg price in canada GE was seen in S839I mice (Fig. 3 C, upper panel) either in the presence or absence of a Cftr inhibitor. However, the extent tab lasix 40mg price in canada 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), tab lasix 40mg price in canada which agrees with the paradoxical observation that upper small intestinal transit is increased in cystic fibrosis patients (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 the increased basal transit rate in S839I mice tab lasix 40mg price in canada was almost solely due to CFTR activation. We then administered linaclotide to more potently activate GC-C (Bryant et al., 2010).
GE was tab lasix 40mg price in canada 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 of mammals, albeit at low levels (Date et tab lasix 40mg price in canada 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 tab lasix 40mg price in canada mice (Fig. 3 D, lower panel). However, transit was significantly higher in S839I mice. Therefore, ligand-mediated activation of hyperactive GC-C causes more tab lasix 40mg price in canada 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) tab lasix 40mg price in canada. 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 tab lasix 40mg price in canada 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) tab lasix 40mg price in canada. 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) tab lasix 40mg price in canada. 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 sodium secretory diarrhea (Müller et al., 2016) tab lasix 40mg price in canada. 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 tab lasix 40mg price in canada 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, tab lasix 40mg price in canada lower panel). In S839I mice, the already elevated basal transit was further enhanced by tenapanor treatment (Fig. 3 I). We attribute this enhanced increase in small intestinal transit to the prevalent lower levels of Nhe3 present in S839I tab lasix 40mg price in canada 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 tab lasix 40mg price in canada al., 2012. Müller et al., 2016). Administration of DSS to mice causes death of tab lasix 40mg price in canada 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 tab lasix 40mg price in canada higher disease activity index (Fig. 4 A, right panel). Colonic shortening was observed in both wild type and S839I tab lasix 40mg price in canada 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., tab lasix 40mg price in canada 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) tab lasix 40mg price in canada. 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 colonic mucosa was observed in S839I mice tab lasix 40mg price in canada (Fig. 4 D). Concomitant with greater mucosal damage, fecal lipocalin, a sensitive marker for inflammation in the gut (Chassaing et tab lasix 40mg price in canada 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 tab lasix 40mg price in canada knockout 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 sodium ion concentrations in the gut lumen may tab lasix 40mg price in canada 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 Nod2â/âCybbâ/â 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 â2 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). Nhe3â/â 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, Il10â/â 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 Crohnâs-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 hormoneâinduced 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 Nhe3â/â 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 Stat1â/â mice following DSS treatment (Bandyopadhyay et al., 2008), and an Ido1â/â 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 â¼24% 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 (5â²-TGAâACAâGTAâCCCâAGGâAGAâTTAâGG-3â²) and Gucy2c_28 (5â²-AACâAGTâTGCâAGAâATCâCTTâGAGâG-3â²) 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 â¼5 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 â¼5 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 â160 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 (12â14 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 antiâβ-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 (7â8 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, 8â12-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 segmentÃsegment 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 manufacturerâs 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 à 300 bp) on the Illumina MiSeq platform.
The V3-V4 hypervariable regions of the 16S rRNA genes were amplified using the 341F (5â²-CCTACGGGNGGCWGCAG-3â²) and 805R (5â²-GACTACHVGGGTATCTAATCC-3â²) 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 * [Ã/sum(Ã)]). 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 KruskalâWallis 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 KruskalâWallis 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:.
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Today, in recognition of National tab lasix 40mg price in canada Womenâs Health Week, and thanks to the American Rescue Plan, the U.S. Department of Health and Human Services (HHS), through the Health Resources and Services Administration (HRSA), awarded approximately $40 million in emergency home visiting funds to states, territories, and the District of Columbia to support children and families affected by the hypertension medications lasix. The Maternal, Infant, and Early Childhood tab lasix 40mg price in canada Home Visiting (MIECHV) Program supports the delivery of coordinated and comprehensive, high quality, voluntary, evidence-based home visiting services to children and families living in communities at risk for poor maternal and child health outcomes.âTodayâs investment demonstrates the Biden Administrationâs commitment to addressing the needs of pregnant people and families, who have been particularly affected by the hypertension medications lasix,â said HHS Secretary Xavier Becerra. ÂIt is essential that we enhance access to home visiting programs so they can support familiesâ crucial health care services, early care and education, and family economic supports.â These funds will be used to provide services and emergency supplies, such as diapers, food, water, and hand sanitizer. Families who cannot access home visiting services due to the lasix will tab lasix 40mg price in canada be provided technology to participate in virtual home visits.
Funds will also be used to train home visitors on emergency preparedness and response planning for families and on how to safely conduct virtual intimate partner violence screenings. ÂThrough innovative programs like MIECHV, HRSA is tab lasix 40mg price in canada committed to improving health and achieving health equity,â said HRSA Acting Administrator Diana Espinosa. ÂThis funding will help bolster evidence-based programs and services that can be a lifeline for low-income parents and families in communities across the country.â The MIECHV Program is administered by HRSA, in partnership with the Administration for Children and Families, to assist underserved parents and families at critical points in their lives. Over the past nine years, tab lasix 40mg price in canada it has provided nearly seven million home visits. In FY 2020, almost three-fourths of families participating in the program had household incomes at or below 100 percent of the Federal Poverty Level, two-thirds of adult participants had a high school education or less and 78 percent of adults and children relied on Medicaid or the Childrenâs Health Insurance Program.
For a list of MIECHV award recipients, visit tab lasix 40mg price in canada. Https://mchb.hrsa.gov/maternal-child-health-initiatives/home-visiting/american-rescue-plan-awards. For more information on HRSA's tab lasix 40mg price in canada Home Visiting Program, visit. Http://mchb.hrsa.gov/programs/homevisiting..
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Recently, 75-year-old Pat had trouble assembling her 40mg lasix twice a day new stationary bike. An avid cyclist in her younger years, she now pedals for 40mg lasix twice a day health and wellness. VOA Services Coordinator Pamela Galloway, center, and nursing students who worked with senior residents.
(Photo taken before current hypertension medications rules.)Who 40mg lasix twice a day did Pat turn to for help with the exercise equipment?. An enthusiast nursing student named Atif Uraizee participating in a community outreach program being pioneered by the Betty Irene Moore School of Nursing at UC Davis."When I stopped by to visit her, she welcomed me into her home, discussed her health, everyday routine and shared personal stories of her life,â Uraizee recalled. "I helped adjust and tighten her new bike and she now includes this in her daily regimen.âPat is one of 80 residents of Rolling 40mg lasix twice a day Oaks, an independent housing community run by the Volunteers of America (VOA).
Uraizee is one of eight graduate nursing students who spent 90 hours this summer 40mg lasix twice a day in clinical rotations at Rolling Oaks working with residents as part of a community health course.âThey are eight extra pairs of eyes and ears and hearts that are listening, hearing, giving their best recommendations and consulting with our team,â said Pamela Galloway, the VOA service coordinator at Rolling Oaks. ÂBeyond the comfort zoneThe idea of nursing interns grew out of a Housing and Urban Development initiative in 2017. The following year, she partnered with the Masterâs Entry in Nursing Program at UC Davis, receiving eight students every 40mg lasix twice a day year to work with residents.
(Nursing students from Sacramento State rotate during other parts of the year.)âI wanted to bring in health and wellness education in a fun way. I thought we could disguise it as a social adventure with these young energetic students,â Galloway said.Roughly 75% of the residents participate in one-on-one visits with the students and group activities to promote health and decrease social isolation.âWeâre hoping residents spend the majority of their time outside of the acute-care setting,â explained Janna Le Page, a UC Davis Health registered 40mg lasix twice a day nurse and clinical instructor. ÂFor these students, their training up to this point is 40mg lasix twice a day acute.
Itâs very task oriented. In this setting, a 40mg lasix twice a day lot of them are outside of their comfort zone. My goal is that they realize a lot of the concepts they learn in community health can transfer to acute care and vice versa.âRecognized for innovationWhile students learn about the world outside a hospital setting, word of the program has reached a national audience.
In August, the American Association of Service Coordinators (AASC) honored Rolling Oaks with the "affordable housing innovative program of the year." The associationâs annual award recognizes affordable 40mg lasix twice a day housing professionals working with vulnerable populations.Most of Rolling Oaks seniors lack support from family. Being a part of this community enhances their quality of life and enables them to live independently and longer in their own homes. During the quarter, the nursing students also conducted a windshield 40mg lasix twice a day survey.
They drove around Rocklin to become familiar with the community, its parks, grocery stores, health care options, transportation services and social supports."Meeting them where theyâre at showed me the importance of seeing people holistically to determine what they need and what resources they have."â Sierra Jensen, nursing studentThe result changed how students viewed the residents and 40mg lasix twice a day the lens through which they provided care.âThis really spoke to the importance of getting to know your patientsâ reality. Asking difficult questions had been hard for me in the acute-care setting but being in their homes made me a lot more comfortable and able to build a rapport,â said MEPN student Sierra Jensen. ÂMeeting them where theyâre at showed me the importance of seeing people holistically to determine what they need and what resources they have.âExpanding a program to connect communities"Since gaining the national spotlight, Galloway has received inquiries from other service coordinators around the country wanting to visit and see the Rolling Oaks 40mg lasix twice a day program firsthand."âMy goal was to see if we can be successful at Rolling Oaks then come up with a template to then put this program into our other VOA senior communities in Placer County and even expand it beyond that,â Galloway said.
ÂNow that weâve been recognized nationally, the connections we've made can benefit everybody.âIt can benefit more residents.âThe students brought joy, good energy and happiness to me,â said resident Sandra Petersen. Â[Their] real concerns made me feel better when they were 40mg lasix twice a day here.âAnd it will benefit the future nurses who graduate in December.âBy going through this, I have a much better idea of what it means to be a community nurse,â Uraizee said. ÂYouâre connecting all the resources and tools, educating and advocating for your patients, and trying to get them the best services out there for better outcomes.â.
Recently, 75-year-old http://andreabroaddus.com/?p=1 Pat tab lasix 40mg price in canada had trouble assembling her new stationary bike. An avid cyclist in her younger years, she now pedals tab lasix 40mg price in canada for health and wellness. VOA Services Coordinator Pamela Galloway, center, and nursing students who worked with senior residents.
(Photo taken before current hypertension medications rules.)Who did Pat turn to tab lasix 40mg price in canada for help with the exercise equipment?. An enthusiast nursing student named Atif Uraizee participating in a community outreach program being pioneered by the Betty Irene Moore School of Nursing at UC Davis."When I stopped by to visit her, she welcomed me into her home, discussed her health, everyday routine and shared personal stories of her life,â Uraizee recalled. "I helped adjust and tighten her new bike and she now includes this in her tab lasix 40mg price in canada daily regimen.âPat is one of 80 residents of Rolling Oaks, an independent housing community run by the Volunteers of America (VOA).
Uraizee is one of eight graduate tab lasix 40mg price in canada nursing students who spent 90 hours this summer in clinical rotations at Rolling Oaks working with residents as part of a community health course.âThey are eight extra pairs of eyes and ears and hearts that are listening, hearing, giving their best recommendations and consulting with our team,â said Pamela Galloway, the VOA service coordinator at Rolling Oaks. ÂBeyond the comfort zoneThe idea of nursing interns grew out of a Housing and Urban Development initiative in 2017. The following year, tab lasix 40mg price in canada she partnered with the Masterâs Entry in Nursing Program at UC Davis, receiving eight students every year to work with residents.
(Nursing students from Sacramento State rotate during other parts of the year.)âI wanted to bring in health and wellness education in a fun way. I thought we could disguise it as a social adventure with these young energetic students,â Galloway said.Roughly 75% of the residents participate tab lasix 40mg price in canada in one-on-one visits with the students and group activities to promote health and decrease social isolation.âWeâre hoping residents spend the majority of their time outside of the acute-care setting,â explained Janna Le Page, a UC Davis Health registered nurse and clinical instructor. ÂFor these students, their training up tab lasix 40mg price in canada to this point is acute.
Itâs very http://msalbasclass.com/2017/03/2252/ task oriented. In this tab lasix 40mg price in canada setting, a lot of them are outside of their comfort zone. My goal is that they realize a lot of the concepts they learn in community health can transfer to acute care and vice versa.âRecognized for innovationWhile students learn about the world outside a hospital setting, word of the program has reached a national audience.
In August, the American Association tab lasix 40mg price in canada of Service Coordinators (AASC) honored Rolling Oaks with the "affordable housing innovative program of the year." The associationâs annual award recognizes affordable housing professionals working with vulnerable populations.Most of Rolling Oaks seniors lack support from family. Being a part of this community enhances their quality of life and enables them to live independently and longer in their own homes. During the quarter, the nursing tab lasix 40mg price in canada students also conducted a windshield survey.
They drove around Rocklin to become familiar with the tab lasix 40mg price in canada community, its parks, grocery stores, health care options, transportation services and social supports."Meeting them where theyâre at showed me the importance of seeing people holistically to determine what they need and what resources they have."â Sierra Jensen, nursing studentThe result changed how students viewed the residents and the lens through which they provided care.âThis really spoke to the importance of getting to know your patientsâ reality. Asking difficult questions had been hard for me in the acute-care setting but being in their homes made me a lot more comfortable and able to build a rapport,â said MEPN student Sierra Jensen. ÂMeeting them where theyâre at showed me the importance of seeing people holistically to determine what they need and what resources they have.âExpanding a program to connect communities"Since gaining the national spotlight, Galloway has received inquiries from other service coordinators around the country wanting to visit and see the Rolling Oaks program firsthand."âMy goal was to see if we can be successful at Rolling tab lasix 40mg price in canada Oaks then come up with a template to then put this program into our other VOA senior communities in Placer County and even expand it beyond that,â Galloway said.
ÂNow that weâve been recognized nationally, the connections we've made can benefit everybody.âIt can benefit more residents.âThe students brought joy, good energy and happiness to me,â said resident Sandra Petersen. Â[Their] real concerns made me feel better when they were here.âAnd it will benefit the future nurses who graduate in December.âBy going through this, I have a much better idea of what it means to be a community tab lasix 40mg price in canada nurse,â Uraizee said. ÂYouâre connecting all the resources and tools, educating and advocating for your patients, and trying to get them the best services out there for better outcomes.â.
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Posttraumatic stress disorder (PTSD) is a complex psychiatric lasix 40mg para que sirve disorder brought on by physical and/or psychological trauma. How its symptoms, including anxiety, depression and cognitive disturbances arise remains incompletely understood and unpredictable. Treatments and outcomes could potentially be improved if lasix 40mg para que sirve doctors could better predict who would develop PTSD. Now, researchers using magnetic resonance imaging (MRI) have found potential brain biomarkers of PTSD in people with traumatic brain injury (TBI).The study appears in Biological Psychiatry.
Cognitive Neuroscience and Neuroimaging, published by Elsevier."The relationship between TBI and PTSD has garnered increased attention in recent years as studies have shown considerable overlap in risk factors and symptoms," said lead author Murray Stein, MD, MPH, FRCPC, a Distinguished Professor of Psychiatry and Family Medicine &. Public Health at lasix 40mg para que sirve the University of California San Diego, San Diego, La Jolla, CA, USA. "In this study, we were able to use data from TRACK-TBI, a large longitudinal study of patients who present in the Emergency Department with TBIs serious enough to warrant CT (computed tomography) scans."The researchers followed over 400 such TBI patients, assessing them for PTSD at 3 and 6 months after their brain injury. At 3 lasix 40mg para que sirve months, 77 participants, or 18 percent, had likely PTSD.
At 6 months, 70 participants or 16 percent did. All subjects underwent brain imaging after injury."MRI studies conducted within two weeks of injury were used to measure volumes of key structures in the brain thought to be involved in PTSD," said Dr. Stein. "We found that the volume of several of these structures were predictive of PTSD 3-months post-injury."Specifically, smaller volume in brain regions called the cingulate cortex, the superior frontal cortex, and the insula predicted PTSD at 3 months.
The regions are associated with arousal, attention and emotional regulation. The structural imaging did not predict PTSD at 6 months.The findings are in line with previous studies showing smaller volume in several of these brain regions in people with PTSD and studies suggesting that the reduced cortical volume may be a risk factor for developing PTSD. Together, the findings suggest that a "brain reserve," or higher cortical volumes, may provide some resilience against PTSD.Although the biomarker of brain volume differences is not yet robust enough to provide clinical guidance, Dr. Stein said, "it does pave the way for future studies to look even more closely at how these brain regions may contribute to (or protect against) mental health problems such as PTSD."Cameron Carter, MD, Editor of Biological Psychiatry.
Cognitive Neuroscience and Neuroimaging, said of the work, "This very important study uses magnetic resonance imaging to take the field a step closer to understanding why some people develop PTSD after trauma and others do not. It also lays the groundwork for future research aimed at using brain imaging to help predict that a person is at increased risk and may benefit from targeted interventions to reduce the clinical impact of a traumatic event." Story Source. Materials provided by Elsevier. Note.
Content may be edited for style and length..
Posttraumatic stress disorder (PTSD) is a complex tab lasix 40mg price in canada psychiatric disorder brought http://markgrigsby.org/seroquel-online-canada/ on by physical and/or psychological trauma. How its symptoms, including anxiety, depression and cognitive disturbances arise remains incompletely understood and unpredictable. Treatments and outcomes could potentially be improved if doctors could better predict tab lasix 40mg price in canada who would develop PTSD.
Now, researchers using magnetic resonance imaging (MRI) have found potential brain biomarkers of PTSD in people with traumatic brain injury (TBI).The study appears in Biological Psychiatry. Cognitive Neuroscience and Neuroimaging, published by Elsevier."The relationship between TBI and PTSD has garnered increased attention in recent years as studies have shown considerable overlap in risk factors and symptoms," said lead author Murray Stein, MD, MPH, FRCPC, a Distinguished Professor of Psychiatry and Family Medicine &. Public Health at the tab lasix 40mg price in canada University of California San Diego, San Diego, La Jolla, CA, USA.
"In this study, we were able to use data from TRACK-TBI, a large longitudinal study of patients who present in the Emergency Department with TBIs serious enough to warrant CT (computed tomography) scans."The researchers followed over 400 such TBI patients, assessing them for PTSD at 3 and 6 months after their brain injury. At 3 months, 77 participants, or 18 percent, tab lasix 40mg price in canada had likely PTSD. At 6 months, 70 participants or 16 percent did.
All subjects underwent brain imaging after injury."MRI studies conducted within two weeks of injury were used to measure volumes of key structures in the brain thought to be involved in PTSD," said Dr. Stein. "We found that the volume of several of these structures were predictive of PTSD 3-months post-injury."Specifically, smaller volume in brain regions called the cingulate cortex, the superior frontal cortex, and the insula predicted PTSD at 3 months.
The regions are associated with arousal, attention and emotional regulation. The structural imaging did not predict PTSD at 6 months.The findings are in line with previous studies showing smaller volume in several of these brain regions in people with PTSD and studies suggesting that the reduced cortical volume may be a risk factor for developing PTSD. Together, the findings suggest that a "brain reserve," or higher cortical volumes, may provide some resilience against PTSD.Although the biomarker of brain volume differences is not yet robust enough to provide clinical guidance, Dr.
Stein said, "it does pave the way for future studies to look even more closely at how these brain regions may contribute to (or protect against) mental health problems such as PTSD."Cameron Carter, MD, Editor of Biological Psychiatry. Cognitive Neuroscience and Neuroimaging, said of the work, "This very important study uses magnetic resonance imaging to take the field a step closer to understanding why some people develop PTSD after trauma and others do not. It also lays the groundwork for future research aimed at using brain imaging to help predict that a person is at increased risk and may benefit from targeted interventions to reduce the clinical impact of a traumatic event." Story Source.
Materials provided by Elsevier. Note. Content may be edited for style and length..
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