Downregulated In Adenoma Research Articles

A Novel Role of SLC26A3 in Maintenance of Intestinal Barrier Function

Inflammatory Bowel Disease (IBD) is a global health burden currently affecting around 3 million people in the United States with increasing incidence worldwide. The pathogenesis of IBD is still unclear due to the multifactorial nature of the disease. Compromised intestinal barrier is one of the most critical early events linked to the onset of intestinal inflammation. In this regard, SLC26A3 or DRA (Down Regulated in Adenoma) is an essential transporter for chloride absorption in the mammalian intestine and has been identified as an IBD susceptibility gene in certain cohorts. Further, DRA levels are markedly decreased in colonic mucosa of IBD patients and in mouse models of gut inflammation. Also, DRA KO mice are more susceptible to DSS colitis. However, the mechanisms underlying increased susceptibility to inflammation by loss of DRA are not fully understood.AimsTo examine the impact of loss of DRA (in DRA KO mice) on epithelial barrier function and expression of key tight junction and adherens junction proteins and elucidate underlying mechanisms.MethodsWild type and DRA KO mice (8–10 weeks old, M/F) were used. Protein expression was measured by immunoblotting and immunofluorescence. FITC‐dextran flux was examined utilizing Ussing chamber.ResultsDRA KO mice exhibited an increase in colonic paracellular permeability (~ 5 fold, p<0.05) indicating impaired barrier function. This was associated with a decrease in levels of TJ protein ZO‐1 and occludin (~ 65%, p<0.05) and major AJ protein, E‐cadherin (~ 60%, p<0.05). Also, the expression of pore forming, Claudin 2 was significantly upregulated in DRA KO mouse colon (~ 3 fold, p<0.05). No significant changes were observed in claudin 1 or 3. Since mouse intestine is a complex physiological system, we next examined if DRA silencing in a model epithelial Caco‐2 cell line, affects barrier function and tight junction protein expression. shRNA Knock‐down of DRA in Caco‐2 cells resulted in a marked decrease in TEER (~ 40%, p<0.05) and occludin expression (~ 60%, p<0.05). Similarly, enteroids derived from DRA KO mice exhibited decreased expression of occludin and E‐cadherin proteins (~ 50%, p<0.05). There was no alteration in mRNA levels of TJ/AJ proteins in DRA KO mice indicating role of post‐transcriptional mechanisms. To understand the mechanisms underlying these alterations, we next examined the expression of RNA‐binding proteins that are known to regulate the translation of occludin and E‐cadherin. Our results showed a marked increase in expression of CUGBP1 (a negative regulator of TJ/AJ proteins) and a marked decrease in expression of intact HuR (a positive regulator).ConclusionOur studies suggest a novel role of a membrane chloride transporter DRA in maintenance of intestinal barrier function and thus a down regulation of this protein may play a key role in pathogenesis of IBD.Support or Funding InformationSupported by NIDDK and Department of Veterans AffairsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enthalpies of Anion Exchange of Chloride by Pyridinecarboxylate in AV-17-8 Anionite

Enthalpies of Cl– anion exchange for anions of nicotinic, isonicotinic, dipicolinic, and cinchomeronic acids on the strongly basic anionite AV-17-8 are measured at 298 K by means of microcalorimetry. Partial and standard enthalpies of sorption of pyridinecarboxylic acid anions on the Cl-form of AV-17-8 anionite from aqueous solutions are determined. The values of standard enthalpies lie in the range 3–14 kJ/mol, increasing in the order nicotinate, isonicotinate, dipicolinic acid anions, and cinchomeronic acid anions.

Chemical traffic light: A self-calibrating naked-eye sensor for fluoride

We report a low-cost sensing platform for effective naked-eye detection of fluoride ion in aqueous media. The sensor is based on silicon complex of 5,10,15-tritolylcorrole (SiTTCorr) deposited on paper support and designed in a particular way that permits it to perform in a unique sensing event an internal sensor self-calibration and subsequent analysis of fluoride ion in a concentration range from 20 [Formula: see text]g/L to 200 mg/L with a LOD 9 [Formula: see text]g/L, much lower than the WHO guideline value of 1.5 mg/L for fluoride in drinking water. The influence of tetradodecylammonium chloride (TDACl) anion exchanger addition to the performance of SiTTCorr-based sensors was studied and the sensor with optimal ionophore: exchanger [Formula: see text] 2:1 ratio demonstrated the highest sensitivity. The evident color variation of SiTTCorr-based optode from dark pink to intense green occurred upon addition of increasing concentrations of fluoride. A smartphone application equipped with home-written color intensity analysis software as a detector of developed sensor output permitted fluoride content quantification in bottled water and toothpaste samples. Moreover, since at the quantification step the SiTTCorr color variation was significant for the red component of visible light and increase of fluoride content evidently changed this color from red to yellow and then to green, the developed optode was compared to a kind of chemical traffic light, able to detect the presence of fluoride in permitted, borderline or dangerous concentrations, respectively.

Fluorinated Bambusurils as Highly Effective and Selective Transmembrane Cl−/HCO3− Antiporters

Summary The exchange of chloride and bicarbonate across lipid bilayers is an important biological process. Synthetic molecules can act as mobile carriers for these anions, although most show little selectivity. Here we report on three bambus[6]uril macrocycles functionalized with fluorinated benzyl groups, which are able to exchange Cl− and HCO3− efficiently. Remarkably, rates for Cl−/NO3− exchange are two orders of magnitude lower. The higher rates of Cl−/HCO3− transport can be explained by the ability of the bambusurils to complex Cl− and HCO3− simultaneously, facilitating their exchange at the bilayer interface. Furthermore, the exceptionally high affinity and selectivity of these systems for NO3− appear to contribute to the poor Cl−/NO3− exchange. This work not only demonstrates the importance of anion binding characteristics on anion transport but also the potential relevance of bambusurils for anion transport applications considering the high rate observed for Cl−/HCO3− exchange.

Infusion of sodium butyrate promotes rumen papillae growth and enhances expression of genes related to rumen epithelial VFA uptake and metabolism in neonatal twin lambs.

The objective of this study was to evaluate the effect of sodium butyrate (SB) infusion on rumen papillae growth and volatile fatty acid (VFA) uptake and metabolism in neonatal lambs. Seven pairs of newborn twin lambs were used. Within each pair, lambs were assigned to receive an oral infusion of SB at 0.36 g/kg body weight (BW) (SB, n = 7) or the same volume of saline (Con, n = 7). Treatments were administered from 10 to 49 d of age, when all lambs were slaughtered. Results showed that the average daily feed intake (ADFI) of starter, average daily gain (ADG), BW of lambs at ages of 5 and 6 wk in SB group were greater (P < 0.05) than those in Con group. Infusion of SB increased (P < 0.05) the concentrations of acetate, butyrate, and total VFA in the rumen fluid and elevated (P < 0.05) the levels of β-hydroxybutyrate acid (BHBA), insulin-like growth factor-1 (IGF-1), and insulin in plasma. Infusion of SB promoted rumen papillae growth, depicted by higher emptied rumen weight, larger rumen papillae length, width, and surface area, and greater thickness of stratum corneum and total epithelium. Sodium butyrate infusion upregulated (P < 0.05) mRNA expression of cyclin A2, cyclin D1, and cyclin-dependent kinases 6 (CDK6), and downregulated (P < 0.05) mRNA expression of caspase-3 and Bcl-2-associated X protein (Bax) in the rumen epithelia. Moreover, SB infusion also upregulated (P < 0.05) mRNA expression of insulin-like growth factor-1 receptor (IGF-1R), and insulin-like growth factor-binding protein 5 (IGFBP-5), and downregulated (P < 0.05) mRNA expression of insulin-like growth factor-binding protein 3 (IGFBP-3) in the rumen epithelia. Sodium butyrate infusion also enhanced (P < 0.05) gene expressions of monocarboxylate transporter isoform 1 (MCT1), downregulated in adenoma (DRA), 3-hydroxy-3-methylglutaryl-CoA synthase isoform 2 (HMGCS2), and 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL), while depressed (P < 0.05) mRNA expression of sodium/proton exchanger isoform 2 (NHE2) in the rumen epithelia. Our results suggest that the SB infusion can improve animal performance, promote the ruminal papillae growth, and enhance expression of genes related to ruminal epithelial VFA uptake and metabolism in preweaning twin lambs. These findings provide a better understanding of the molecular mechanism of SB promoting rumen epithelial development and function in preweaning lambs.

Cross-Linked Poly(vinylbenzyl chloride) Anion Exchange Membranes with Long Flexible Multihead for Fuel Cells

To enhance the ionic conductivity and mechanical property of the chemically stable aliphatic anion exchange membranes (AEMs), cross-linked poly(vinylbenzyl chloride) AEMs (PVBC-xQ4) bearing long flexible cross-linker chains with multiple quaternary ammonium (QA) head were prepared via a facile one-step process. The AEMs exhibit high conductivity and excellent mechanical property. The cross-linked AEMs excluding any other heteroatomic bond especially the sulfone linkage or ether bond in the backbone have robust alkaline stability, low water uptake, and good dimensional stability. A well-developed hydrophilic/hydrophobic phase separated morphology was observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The PVBC-50Q4 membrane with ionic exchange capacity (IEC) of 1.87 mequiv g–1 displays a highest ionic conductivity of 74.3 mS cm–1 at 80 °C. After tested in a 2 M aqueous KOH solution at 80 °C for 1300 h, 88% of the conductivity and 89% of the IEC were remained. Furthermore, ...

N,N,N-Trimethyl-5-[(2,3,5,6-tetrafluorophenoxy)carbonyl]pyridin-2-aminium trifluoromethanesulfonate a precursor for the synthesis of 2,3,5,6-tetrafluorophenyl 6-[18F]-fluoronicotinate.

The synthesis, recrystallization, and X-ray deterimination of N,N,N-trimethyl-5-[(2,3,5,6-tetrafluorophenoxy)carbonyl]pyridin-2-aminium trifluoromethanesulfonate (PyTFP-precursor), C15H13F4N2O2+·CF3SO3-, is described. This triflate salt precursor is required for the synthesis of 2,3,5,6-tetrafluorophenyl 6-[18F]-fluoronicotinate ([18F]FPyTFP), a prosthetic group used to radiolabel peptides for positron emission tomography (PET), as peptides are increasingly being used as PET-imaging probes in nuclear medicine. Radiolabeling of peptides is typically done using a `prosthetic group', a small synthon to which the radioisotope is attached in the first step, followed by attachment to the peptide in the second step. During the synthesis of the PyTFP-precursor, displacement of a Cl atom with trimethylamine gas and anion replacement with a triflate counter-ion is critical, as incomplete replacement would hinder radioisotopic incorporation of nucleophilic fluorine-18 and result in diminished radiochemical yields. The structural determination of the PyTFP-precursor by X-ray crystallography helped confirm the anion exchange of chloride with triflate.

SLC26A3 (DRA) prevents TNF-alpha-induced barrier dysfunction and dextran sulfate sodium-induced acute colitis

SLC26A3 encodes a Cl−/HCO3− ion transporter that is also known as downregulated in adenoma (DRA) and is involved in HCO3−/mucus formation. The role of DRA in the epithelial barrier has not been previously established. In this study, we investigated the in vivo and in vitro mechanisms of DRA in the colon epithelial barrier. Immunofluorescence (IF) and co-immunoprecipitation (co-IP) studies reveal that DRA binds directly to tight junction (TJ) proteins and affects the expression of TJ proteins in polarized Caco-2BBe cells. Similarly, DRA colocalizes with ZO-1 in the intestinal epithelium. Knockdown or overexpression of DRA leads to alterations in TJ proteins and epithelial permeability. In addition, TNF-α treatment downregulates DRA by activating NF-кB and subsequently affecting intestinal epithelial barrier integrity. Furthermore, overexpression of DRA partly reverses the TNF-α-induced damage by stabilizing TJ proteins. Neutralization of TNF-α in dextran sulfate sodium (DSS)-induced colitis mice demonstrates improved the outcomes, and the therapeutic effect of the TNF-α neutralizing mAb is mediated in part by the preservation of DRA expression. These data suggest that DRA may be one of the therapeutic targets of TNF-α. Moreover, DRA delivered by adenovirus vector significantly prevents the exacerbation of colitis and improves epithelial barrier function by promoting the recovery of TJ proteins in DSS-treated mice. In conclusion, DRA plays a role in protecting the epithelial barrier and may be a therapeutic target in gut homeostasis.

Local Colonic Delivery of Vasoactive Intestinal Peptide (VIP) Nanomedicine Alleviates Colitis in Mice

Inflammatory Bowel Disease (IBD) is an unmet medical need encompassing two entities; Ulcerative Colitis (UC) and Crohn's Disease (CD). We have previously demonstrated the superior therapeutic effects of systemically administered VIP nanomedicine (VIP in sterically stabilized micelles; VIP‐SSM), in alleviating colitis in mice. In addition, we have also characterized a luminally expressed VPAC1 receptor in human and mouse colonic mucosa. Thus, the current studies were undertaken to determine if delivering the nanomedicine directly to the colonic lumen could have similar therapeutic benefits.MethodsVIP‐SSM was prepared by mixing aqueous solutions of lipid and peptide in a predetermined molar ratio and was characterized for its size by dynamic light scattering. Colitis was induced in C57Bl/6 mice with 3.5% w/v dextran sulfate sodium (DSS) in drinking water for 7 days. Animals were randomly divided into 5 groups; (Control, DSS, VIP‐SSM, DSS+VIP‐SSM and DSS+ VIP). On the 5th day mice were anesthetized with ketamine/xylazine and catheterized to intra rectally instill 100 μls of, VIP‐SSM (0.25 nmol), free VIP (0.25 nmol) or SSM (100 nmol). At day 7, mice were switched to tap water and sacrificed at day 10. Body weights of mice were recorded daily. Colon appearance was observed for diarrheal phenotype, distal colonic mucosa was used for RNA and protein isolation for qPCR and western blot analysis, respectively. Frozen sections of colon were used for H &amp; E and immunostaining.ResultsThe prepared nanoparticle hydrodynamic particle sizes were approximately 15 nm. At day 10, VIP‐SSM treated mice showed significant improvement in body weight compared to DSS and free VIP treated DSS mice. The diarrheal phenotype seen with DSS indicated by loose stool in colon and reduced length was alleviated with VIP‐SSM (showing presence of solid fecal pellets) and improved colon length. In parallel, reduced expression of ion transporters such as SLC26a3 or down regulated in adenoma (DRA) seen in colitis was abrogated with VIP‐SSM intra rectal treatment but not with the free VIP peptide. Furthermore, increased pro‐inflammatory cytokine mRNA expression and damaged distal colonic histology and goblet cell loss seen in DSS was also significantly alleviated with VIP‐SSM treatment. The VPAC1 receptor expression was similar across all groups.ConclusionsThese results demonstrate that local administration of VIP nanomedicine is effective in alleviating severe inflammation and associated diarrhea in colitis. Thus, delivering VIP‐SSM nanoparticle orally with site‐specific release to the colon could be a potential option in managing UC in future.Support or Funding InformationNIH/NIDDK, Department of Veteran AffairsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dietary supplements during the cold season increase rumen microbial abundance and improve rumen epithelium development in Tibetan sheep.

Livestock on the Qinghai-Tibetan Plateau are faced with extreme harsh winters and are often in negative energy balance during this period. Dietary supplementation can improve growth performance of Tibetan sheep and, consequently, we hypothesized that it would also increase microbial abundance and rumen epithelium development. To test this hypothesis, we examined the effect of feed supplementation during the cold season on rumen microbes, fermentation, epithelium development, and absorptive capability in Tibetan sheep. Eighteen 1-yr-old ewes (BW = 29.4 ± 1.79, kg) were offered oat hay ad libitum for 60 d and divided randomly into three groups: 1) no supplement; control group (CON); 2) urea-molasses lick block supplement (BS); and 3) concentrate feed supplement (CS). The ADG of CS ewes (143.3, g/d) was greater (P < 0.05) than BS ewes (87.9, g/d), which was greater (P < 0.05) than CON ewes (44.5, g/d). Serum concentrations of GH, IGF-1, and IGF-2 in the CS and BS groups were greater than in the CON group (P < 0.05). Greater relative abundance of protozoa, Ruminococcus albus, Fibrobacter succinogenes, Streptococcus bovis, and Ruminobacter amylophilus was observed in the CS and BS groups than in the CON group (P < 0.05), and relative abundances of rumen fungi, Butyrivibrio fibrisolvens, and Prevotella ruminicola in the CS group were greater than in the BS and CON groups (P < 0.05). Ruminal total VFA, ammonia, and microbial protein concentrations in the CS and BS groups were greater than in the CON group (P < 0.05), and in the CS group were greater than in the BS group (P < 0.05). Ruminal papillae width and surface area in the CS and BS groups were greater than in the CON group (P < 0.05), while in the CS group were greater than in the BS group (P < 0.05). The mRNA expressions of IGFBP5, NHE1 (sodium/hydrogen antiporter, isoform 1), DRA (downregulated in adenoma), and Na+/K+-ATPase (sodium/potassium ATPase pump) in ruminal epithelium were greater in the CS and BS groups than in the CON group (P < 0.05), and in the CS group was greater than in the BS group (P < 0.05), while NHE3 (sodium/hydrogen antiporter, isoform 3), MCT1 (monocarboxylate transporter 1), and MCT4 (monocarboxylate transporter 4) mRNA expressions in the CS group were greater than in the BS and CON groups (P < 0.05). It was concluded that supplementing Tibetan sheep during the cold season increases rumen microbial abundance and improves fermentation parameters, rumen epithelium development, and absorptive capability.

Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers

Background & AimsHuman enteroids present a novel tool to study human intestinal ion transport physiology and pathophysiology. The present study describes the contributions of Cl- and HCO3- secretion to total cyclic adenosine monophosphate (cAMP)-stimulated electrogenic anion secretion in human duodenal enteroid monolayers and the relevant changes after differentiation.MethodsHuman duodenal enteroids derived from 4 donors were grown as monolayers and differentiated by a protocol that includes the removal of Wnt3A, R-spondin1, and SB202190 for 5 days. The messenger RNA level and protein expression of selected ion transporters and carbonic anhydrase isoforms were determined by quantitative real-time polymerase chain reaction and immunoblotting, respectively. Undifferentiated and differentiated enteroid monolayers were mounted in the Ussing chamber/voltage-current clamp apparatus, using solutions that contained as well as lacked Cl- and HCO3-/CO2, to determine the magnitude of forskolin-induced short-circuit current change and its sensitivity to specific inhibitors that target selected ion transporters and carbonic anhydrase(s).ResultsDifferentiation resulted in a significant reduction in the messenger RNA level and protein expression of cystic fibrosis transmembrane conductance regulator, (CFTR) Na+/K+/2Cl- co-transporter 1 (NKCC1), and potassium channel, voltage gated, subfamily E, regulatory subunit 3 (KCNE3); and, conversely, increase of down-regulated-in-adenoma (DRA), electrogenic Na+/HCO3- co-transporter 1 (NBCe1), carbonic anhydrase 2 (CA2), and carbonic anhydrase 4 (CA4). Both undifferentiated and differentiated enteroids showed active cAMP-stimulated anion secretion that included both Cl- and HCO3- secretion as the magnitude of total active anion secretion was reduced after the removal of extracellular Cl- or HCO3-/CO2. The magnitude of total anion secretion in differentiated enteroids was approximately 33% of that in undifferentiated enteroids, primarily owing to the reduction in Cl- secretion with no significant change in HCO3- secretion. Anion secretion was consistently lower but detectable in differentiated enteroids compared with undifferentiated enteroids in the absence of extracellular Cl- or HCO3-/CO2. Inhibiting CFTR, NKCC1, carbonic anhydrase(s), cAMP-activated K+ channel(s), and Na+/K+-adenosine triphosphatase reduced cAMP-stimulated anion secretion in both undifferentiated and differentiated enteroids.ConclusionsHuman enteroids recapitulate anion secretion physiology of small intestinal epithelium. Enteroid differentiation is associated with significant alterations in the expression of several ion transporters and carbonic anhydrase isoforms, leading to a reduced but preserved anion secretory phenotype owing to markedly reduced Cl- secretion but no significant change in HCO3- secretion.

Quaternized poly(styrene-co-vinylbenzyl chloride) anion exchange membranes: role of different ammonium cations on structural, morphological, thermal and physio-chemical properties

Investigating the effect of various ammonium cations on poly(styrene-co-vinylbenzyl chloride) based anion exchange membranes and correlating their structure–property relationship.

Tumor necrosis factor-α acts reciprocally with solute carrier family 26, member 3, (downregulated-in-adenoma) and reduces its expression, leading to intestinal inflammation.

Solute carrier family 26, member 3 (Slc26a3), also termed downregulated-in-adenoma (DRA) is a member of the Slc26 family of anion transporters and is mutated in congenital chloride diarrhea. Our previous study demonstrated that DRA deficiency is associated with severely reduced colonic HCO3− secretion, a loss of colonic fluid absorption, a lack of a firmly adherent mucus layer and a severely reduced colonic mucosal resistance to dextran sodium sulfate (DSS) damage. However, the direct effect of mediators that trigger intestinal inflammatory factors on DRA has not been fully investigated. Tumor necrosis factor (TNF)-α is a central mediator of intestinal inflammation in inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease. However, to the best of our knowledge, whether TNF-α acts reciprocally with DRA leading to the development of gut inflammation in IBD has not been reported. The present study identified that the expression level of DRA was reduced in active UC patients and DSS-induced colitis mice with high expression levels of TNF-α identified in the peripheral blood serum. In addition, TNF-α may affect the expression level of DRA in human colonic Caco2BBE cells in a dose-dependent manner, including in DRA overexpressed Caco2BBE cells. Furthermore, knockdown of TNF-α in Caco2BBE cells led to a higher expression level of DRA and a markedly reduced secretion of TNF-α in the culture media. In addition, knockdown of DRA in Caco2BBE cells led to a higher secretion of TNF-α in the culture media compared with the control cells, which could be reversed by overexpression of DRA. Overall, these results indicate that TNF-α may act reciprocally with DRA, leading to the development of intestinal inflammation. Based on the pivotal position of TNF-α in IBD, DRA is hypothesized to have therapeutic potential against colitis serving as an important target.

Vasoactive Intestinal Peptide Nanomedicine for the Management of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic relapsing disorder of the intestine, with increasing incidence worldwide. At present, the management of IBD is an unmet medical need due to the ineffectiveness of currently available drugs in treating all patients, and there is strong demand for novel therapeutics. In this regard, vasoactive intestinal peptide, a potent anti-inflammatory endogenous hormone, has shown promise in managing multiple immune disorders in animal models. However, when administered in the free form, VIP undergoes rapid degradation in vivo, and with continuous infusion, it causes severe dose limiting side effects. To overcome these barriers, we have developed a superior mode to deliver VIP in its native form, using sterically stabilized micelles (VIP-SSM). Our previous studies demonstrated that, VIP, when administered in SSM, prevented joint damage and inflammation in a mouse model of rheumatoid arthritis at a significantly lower dose than the free peptide, completely abrogating the serious side effect of hypotension associated with VIP. In the current study, we demonstrate the therapeutic benefit of VIP-SSM over free peptide in reversing severe colitis associated with IBD. First, we conducted preliminary studies with dextran sulfate sodium (DSS) induced colitis in mice, to determine the effectiveness of VIP administered on alternate days in reducing disease severity. Thereafter, a single intra peritoneal injection of VIP-SSM or the free peptide was used to determine its therapeutic effect on the reversal of colitis and associated diarrhea. The results demonstrated that when administered on alternate days, both VIP-SSM and VIP were capable of alleviating DSS colitis in mice. However, when administered as a single dose, in a therapeutic setting, VIP-SSM showed superior benefits compared to the free peptide in ameliorating colitis phenotype. Namely, the loss of solid fecal pellets and increased fluid accumulation in colon resulting from DSS insult was abrogated in VIP-SSM treated mice and not with free VIP. Furthermore, reduced protein and mRNA levels of the major chloride bicarbonate exchanger, down regulated in adenoma (DRA), seen with DSS was reversed with VIP-SSM, but not with the free peptide. Similarly, VIP-SSM treatment significantly reduced the elevated mRNA levels of pro-inflammatory cytokines and showed significant histologic recovery when compared to mice treated with free VIP. Therefore, these results demonstrated that as a single dose, the anti-inflammatory and antidiarrheal effects of VIP can be achieved effectively when administered as a nanomedicine. Therefore, we propose VIP-SSM to be developed as a potential therapeutic tool for treating ulcerative colitis, a type of IBD.

Synthesis of polyketone-g-vinylbenzyl chloride anion exchange membrane via irradiation and its properties

A novel anion exchange membrane composed of aminated polyketone-g-vinylbenzyl chloride was synthesized via 60Co γ-ray irradiation grafting technique. Total irradiation dose and concentration of monomers were set as variables to determine degree of grafting. Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy equipped with energy dispersive X-ray spectrometer (SEM-EDS) were used to characterize the grafted membranes. Water uptake (WU) and swelling ratio (SR) were measured with a gravimetric method. A titration method was used to detect ion exchange capacity (IEC), whereas electrical resistance (ER) and ion conductivity (IC) were measured with a LCR meter. Our method has boosted the degree of grafting up to 97.6% from 3.7% as total irradiation dose and monomer concentration have been increased. WU and SR were in the ranges 1.5%-35.6% and 0.35%-20.9%, respectively. Also, IEC has taken values ranging between 0.2 and 1.19 meq/g. IC has varied within the range 0.01-0.3 S cm-1, and it has shown a rising trend as IEC increased. In this study, the optimum synthesis conditions have been 70 kGy of total irradiation dose and 50wt% of monomers. The membrane synthesized here is potentially superior to Nafion membrane in terms of several properties. Open image in new window

CDX2 upregulates SLC26A3 gene expression in intestinal epithelial cells.

SLC26A3 [downregulated in adenoma (DRA)] plays a key role in mammalian intestinal NaCl absorption, in that it mediates apical membrane Cl-/[Formula: see text] exchange. DRA function and expression are significantly decreased in diarrhea associated with inflammatory bowel disease. DRA is also considered to be a marker of cellular differentiation and is predominantly expressed in differentiated epithelial cells. Caudal-type homeobox protein-2 (CDX2) is known to regulate genes involved in intestinal epithelial differentiation and proliferation. Reduced expression of both DRA and CDX2 in intestinal inflammation prompted us to study whether the DRA gene is directly regulated by CDX2. Our initial studies utilizing CDX2 knockout (CDX2fV/fV;Cre+) mice showed a marked reduction in DRA mRNA and protein levels in proximal and distal colon. In silico analysis of the DRA promoter showed two consensus sites for CDX2 binding. Therefore, we utilized Caco-2 cells as an in vitro model to examine if DRA is a direct target of CDX2 regulation. siRNA-mediated silencing of CDX2 in Caco-2 cells resulted in a marked (~50%) decrease in DRA mRNA and protein levels, whereas ectopic overexpression of CDX2 upregulated DRA expression and also stimulated DRA promoter activity, suggesting transcriptional regulation. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated direct binding of CDX2 to one of the two putative CDX2 binding sites in the DRA promoter (+645/+663). In summary, our studies, for the first time, demonstrate transcriptional regulation of DRA expression by CDX2, implying that reduced expression of DRA in inflammatory bowel disease-associated diarrhea may, in part, be due to downregulation of CDX2 in the inflamed mucosa.NEW & NOTEWORTHY SLC26A3 [downregulated in adenoma (DRA)] mediates intestinal luminal NaCl absorption and is downregulated in inflammatory bowel disease-associated diarrhea. Since both DRA and caudal-type homeobox protein-2 (CDX2) are reduced in intestinal inflammation and the DRA promoter harbors CDX2 binding sites, we examined whether the DRA gene is regulated by CDX2. Our studies, for the first time, demonstrate transcriptional regulation of DRA expression by CDX2 via direct binding to the DRA promoter, suggesting that reduced expression of DRA in inflammatory bowel disease-associated diarrhea could, in part, be attributed to downregulation of CDX2.

Loss of the anion exchanger DRA (Slc26a3), or PAT1 (Slc26a6), alters sulfate transport by the distal ileum and overall sulfate homeostasis.

The ileum is considered the primary site of inorganic sulfate ([Formula: see text]) absorption. In the present study, we explored the contributions of the apical chloride/bicarbonate (Cl-/[Formula: see text]) exchangers downregulated in adenoma (DRA; Slc26a3), and putative anion transporter 1 (PAT1; Slc26a6), to the underlying transport mechanism. Transepithelial 35[Formula: see text] and 36Cl- fluxes were determined across isolated, short-circuited segments of the distal ileum from wild-type (WT), DRA-knockout (KO), and PAT1-KO mice, together with measurements of urine and plasma sulfate. The WT distal ileum supported net sulfate absorption [197.37 ± 13.61 (SE) nmol·cm-2·h-1], but neither DRA nor PAT1 directly contributed to the unidirectional mucosal-to-serosal flux ([Formula: see text]), which was sensitive to serosal (but not mucosal) DIDS, dependent on Cl-, and regulated by cAMP. However, the absence of DRA significantly enhanced net sulfate absorption by one-third via a simultaneous rise in [Formula: see text] and a 30% reduction to the secretory serosal-to-mucosal flux ([Formula: see text]). We propose that DRA, together with PAT1, contributes to [Formula: see text] by mediating sulfate efflux across the apical membrane. Associated with increased ileal sulfate absorption in vitro, plasma sulfate was 61% greater, and urinary sulfate excretion (USO4) 2.2-fold higher, in DRA-KO mice compared with WT controls, whereas USO4 was increased 1.8-fold in PAT1-KO mice. These alterations to sulfate homeostasis could not be accounted for by any changes to renal sulfate handling suggesting that the source of this additional sulfate was intestinal. In summary, we characterized transepithelial sulfate fluxes across the mouse distal ileum demonstrating that DRA (and to a lesser extent, PAT1) secretes sulfate with significant implications for intestinal sulfate absorption and overall homeostasis.NEW & NOTEWORTHY Sulfate is an essential anion that is actively absorbed from the small intestine involving members of the Slc26 gene family. Here, we show that the main intestinal chloride transporter Slc26a3, known as downregulated in adenoma (DRA), also handles sulfate and contributes to its secretion into the lumen. In the absence of functional DRA (as in the disease congenital chloride diarrhea), net intestinal sulfate absorption was significantly enhanced resulting in substantial alterations to overall sulfate homeostasis.

A new synthesis of N-alkoxyaminopyridinium salts

It was found, that methyl N-alkoxy-N-chlorocarbamates interaction with 4-dimethylaminopyridine was overall manner of synthesis of 1-N-alkoxyaminopyridinium chlorides. New1-N-n-octyloxyamino-4-dimethylaminopyridinium and 1-N-benzyloxyamino-4-dimethylaminopyridinium chlorides have been synthesized by the methyl N-chloro-N-n-octyloxycarbamate and methyl N-benzyloxy-N-chlorocarbamate interaction with 4-dimethylaminopyridine in acetonitrile solution, respectively. The novel synthesis of 1-N-alkoxyaminopyridinium chlorides by reaction of N- -алкоксимочевин alkoxy-N-(1-pyridinium)ureas chlorides ammonolysis has been proposed. 1-N-Methoxyamino-4-dimethylaminopyridinium chloride and 1-N-n-butyloxyamino-4-dimethylaminopyridinium chloride have been synthesized by reaction of N-1-4-dimethylaminopyridinium-N-methoxyurea chloride and N-n-butyloxy-N-(1-4-dimethylaminopyridinium)urea chloride, respectively, with excess of ammonia in ethanol solution at room temperature during few minutes. Evidently, the carbamoyl proton elimination by ammonia causes further formation 1-N-alkoxy-4-dimethylaminopyridinium chlorides and HNCO. It is new example of decarbamoylation of N-alkoxy-N-(1-pyridinium)ureas salts. 1-N-Metoxyamino-4-dimethylaminopyridinium fluoride has been synthesized by N-1-(4-dimethylamino)pyridinium-N-methoxyurea chloride reaction with potassium fluoride in acetonitrile. Probably, in this reaction N-1-(4-dimethylamino)pyridinium-N-methoxyurea caution decarbamoylation occurs after the chloride anion exchange on fluoride anion. 1-N-Alkoxyaminopyridinium salts structures have been confirmed by data of NMR 1H spectra, NMR 13C spectra and mass-spectra

Reduced Expression of Down‐Regulated in Adenoma (DRA) in Murine Enteroid‐Derived Monolayers Following Infection with Salmonella

Non‐typhoidal Salmonella spp. are the second leading cause of foodborne illness worldwide. Despite its prevalence, the mechanism through which Salmonella infection causes diarrhea is not fully understood. The intestinal epithelium plays a major role in both interacting with bacteria and fluid accumulation. To understand the basic mechanism of Salmonella infection in intestinal epithelial cells, we developed a murine enteroid model that mimics the crypt‐villus axis of the intestines, and contains the four different epithelial subtypes found in the intestine (enterocytes, enteroendocrine, goblet, and Paneth cells). We have previously shown that infection with Salmonella causes decreased expression of the chloride/bicarbonate exchanger SLC26A3 (DRA; Downregulated in Adenoma) in the murine colon. We developed enteroid‐derived monolayers (EDM) from mouse ileum and colon to test whether DRA was similarly down‐regulated following infection with Salmonella. Monolayers were generated from enteroids on either permeable transepithelial supports or cell culture plates. Monolayers were also grown in 8‐well chamber slides to visualize DRA and bacterial internalization. After differentiation, EDM were infected with Salmonella enterica serovar Typhimurium. Expression of DRA and markers for different cell subtypes were studied using RT‐PCR, western blotting, and confocal microscopy. We also measured bacterial internalization using a gentamicin protection assay followed by colony counts. We found bacterial internalization after 1 h followed by decreased expression of DRA after 6 h of Salmonella infection. Interestingly, following infection the proliferation marker Ki67 was upregulated. The intestines contain both secretory (enteroendocrine, goblet, and paneth cells) and absorptive (enterocytes) cell types and their expression was evaluated following infection. The Wnt/NOTCH signaling pathways control cell fate decisions in the intestines. We found that Hes1, a downstream signaling molecule in the NOTCH signaling pathway, was up regulated following infection. Our work shows the potential for EDM to identify molecular mechanisms that may underlie intestinal dysfunction during infection. Ultimately, this model may identify signals that can be targeted for therapeutic development.

Novel process combining anaerobic-aerobic digestion and ion exchange resin for full recycling of cassava stillage in ethanol fermentation

A novel cleaner ethanol production process has been developed. Thin stillage is treated initially by anaerobic digestion followed by aerobic digestion and then further treated by chloride anion exchange resin. This allows the fully-digested and resin-treated stillage to be completely recycled for use as process water in the next ethanol fermentation batch, which eliminates wastewater discharges and minimizes consumption of fresh water. The method was evaluated at the laboratory scale. Process parameters were very similar to those found using tap water. Maximal ethanol production rate in the fully-recycled stillage was 0.9g/L/h, which was similar to the 0.9g/L/h found with the tap water control. The consumption of fresh water was reduced from 4.1L/L (fresh water/ethanol) to zero. Compared with anaerobically-aerobically digested stillage which had not been treated with resin, the fermentation time was reduced by 28% (from 72h to 52h) and reached the level achieved with tap water. This novel process can assist in sustainable development of the ethanol industry.