Rabbit Ileum Research Articles

RhoA-mediated Ca2+ Sensitization in Erectile Function

A Rho-kinase inhibitor increases corpus cavernosum (CC) pressure in an in vivo rat model (Chitaley, K., Wingard, C. J., Webb, R. C., Branam, H., Stopper, V. S., Lewis, R. W., and Mills, T. M. (2001) Nat. Med. 7, 119-122) suggesting that Rho-mediated Ca(2+) sensitization of CC smooth muscle maintains the flaccid (contracted) state. We directly demonstrate Ca(2+) sensitization of permeabilized rabbit and human CC and identify a highly expressed molecular component of this pathway. Ca(2+) sensitization of force induced by endothelin or GTPgammaS was significantly greater in CC than in rabbit ileum smooth muscle and was accompanied by a 17-fold higher RhoA content. Pull-down assays with the RhoA binding domain of mDia showed the high RhoA content of CC to be available for activation by GTPgammaS. Ca(2+) sensitization induced by endothelin, phenylephrine, or GTPgammaS was completely relaxed by the Rho kinase inhibitor Y-27632. Human and rabbit CC both express the phosphatase inhibitor CPI-17, the myosin phosphatase regulatory (MYPT-1) and catalytic (PP1delta) subunits, and two isoforms of Rho kinase. We suggest that high expression of RhoA contributes, through RhoA-mediated Ca(2+) sensitization, to the flaccid state of CC that can be reversed by a water-soluble, orally active Rho kinase inhibitor suitable for therapy of erectile dysfunction.

Blocking actions of the sea anemone, Bunodosoma cavernata extract on Histamine and Acetylcholine induced contractile actions of the wild grasscutter and rabbit Ilea

Extract from the sea anemone, Bunodosoma cavernata was prepared by homogenizing 100 of the sea anemones in 300ml of 0.9% saline in a Moulinex blender. This was centrifuged at 4000 revolutions per minute for 10 minutes. The supernatant was re – centrifuged to obtain the crude extract. The extract (1ml/l) was found to elicit long lasting contractions on the ilea of wild rabbit and grass cutter. It was also found to block acetylcholine (ACh) – induced contractions of the ileum of grass cutter (90 + 6.2% inhibition + SEM) and the contractile action induced by ACh (0.2mg and 0.4mg) (89 + 5.8% inhibition + SEM) and histamine (0.075mg and 0.15mg) on the rabbit ileum (80 + 4.6% inhibition + SEM) . The antagonism by the extract was non – selective. (Journal of Applied Sciences & Environmental Management: 2002 6(2): 11-16)

Anastomosis in rabbit ileum (IL) and colon (CO) affects smooth muscle contractility and mitochondrial respiratory activity

Anastomosis in rabbit ileum (IL) and colon (CO) affects smooth muscle contractility and mitochondrial respiratory activity

Identification of a Ligand-binding Site in the Na+/Bile Acid Cotransporting Protein from Rabbit Ileum

Reabsorption of bile acids occurs in the terminal ileum by a Na(+)-dependent transport system composed of several subunits of the ileal bile acid transporter (IBAT) and the ileal lipid-binding protein. To identify the bile acid-binding site of the transporter protein IBAT, ileal brush border membrane vesicles from rabbit ileum were photoaffinity labeled with a radioactive 7-azi-derivative of cholyltaurine followed by enrichment of IBAT protein by preparative SDS gel electrophoresis. Enzymatic fragmentation with chymotrypsin yielded IBAT peptide fragments in the molecular range of 20.4-4 kDa. With epitope-specific antibodies generated against the C terminus a peptide of molecular mass of 6.6-7 kDa was identified as the smallest peptide fragment carrying both the C terminus and the covalently attached radiolabeled bile acid derivative. This clearly indicates that the ileal Na(+)/bile acid cotransporting protein IBAT contains a bile acid-binding site within the C-terminal 56-67 amino acids. Based on the seven-transmembrane domain model for IBAT, the bile acid-binding site is localized to a region containing the seventh transmembrane domain and the cytoplasmic C terminus. Alternatively, assuming the nine-transmembrane domain model, this bile acid-binding site is localized to the ninth transmembrane domain and the C terminus.

Decreased apoptosis in the ileum and ileal Peyer's patches: a feature after infection with rabbit enteropathogenic Escherichia coli O103.

Significant changes occur in intestinal epithelial cells after infection with enteropathogenic Escherichia coli (EPEC). However, it is unclear whether this pathogen alters rates of apoptosis. By using a naturally occurring weaned rabbit infection model, we determined physiological levels of apoptosis in rabbit ileum and ileal Peyer's patches (PP) and compared them to those found after infection with adherent rabbit EPEC (REPEC O103). Various REPEC O103 strains were first tested in vitro for characteristic virulence features. Rabbits were then inoculated with the REPEC O103 strains that infected cultured cells the most efficiently. After experimental infection, intestinal samples were examined by light and electron microscopy. Simultaneously, ileal apoptosis was assessed by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and caspase 3 assays and by apoptotic cell counts based on morphology (hematoxylin-and-eosin staining). The highest physiological apoptotic indices were measured in PP germinal centers (median = 14.7%), followed by PP domed villi (8.1%), tips of absorptive villi (3.8%), and ileal crypt regions (0.5%). Severe infection with REPEC O103 resulted in a significant decrease in apoptosis in PP germinal centers (determined by TUNEL assay; P = 0.01), in the tips of ileal absorptive villi (determined by H&E staining; P = 0.04), and in whole ileal cell lysates (determined by caspase 3 assay; P = 0.001). We concluded that REPEC O103 does not promote apoptosis. Furthermore, we cannot rule out the possibility that REPEC O103, in fact, decreases apoptotic levels in the rabbit ileum.

Impairment of contractility in vitro with abnormal mitochondrial response in rabbit ileum (IL) and colon (CO) after anastomosis

Impairment of contractility in vitro with abnormal mitochondrial response in rabbit ileum (IL) and colon (CO) after anastomosis

Impairment of contractility in vitro with abnormal mitochondrial response in rabbit ileum (IL) and colon (CO) after anastomosis

Impairment of contractility in vitro with abnormal mitochondrial response in rabbit ileum (IL) and colon (CO) after anastomosis

Enteropathogenic Escherichia coli Strain RDEC-1 Produces a Novel Electrogenic Factor Active on Rabbit Ileum In Vitro

Attaching and effacing Escherichia coli demonstrate marked species specificity in inducing diarrhea, although its mechanism remains largely unclear. The purpose of this study was to investigate the existence of a soluble, species-specific factor that induces diarrhea in an in vitro model. Stripped rabbit ileum was mounted in Ussing chambers, and changes in potential difference and short-circuit current were monitored after the addition of bacterial culture supernatant. The culture supernatant from rabbit-specific strain RDEC-1, but not from human-specific enteropathogenic Escherichia coli strain E2348/69, induced an increase in potential difference and short-circuit current in rabbit ileum mounted in Ussing chambers. This electrical signal was related to chloride ion secretion, was absent in colonic tissue, and was retained in the 30 to 100-KDa fraction of the supernatant. Preliminary experiments failed to show an involvement of calcium or cyclic nucleotides as intracellular messengers. RDEC-1 cured of a 42-MDa plasmid lost the enterotoxicity whereas conjugation of the plasmid into the negative E. coli recipient HB101 resulted in the expression of toxicity. The authors describe a novel, species-specific factor that helps to explain RDEC-1 diarrhea, which may be relevant to the pathogenesis of enteropathogenic Escherichia coli infection.

Enteropathogenic Escherichia coli Strain RDEC‐1 Produces a Novel Electrogenic Factor Active on Rabbit Ileum In Vitro

ABSTRACTBackgroundAttaching and effacing Escherichia coli demonstrate marked species specificity in inducing diarrhea, although its mechanism remains largely unclear. The purpose of this study was to investigate the existence of a soluble, species‐specific factor that induces diarrhea in an in vitro model.MethodsStripped rabbit ileum was mounted in Ussing chambers, and changes in potential difference and short‐circuit current were monitored after the addition of bacterial culture supernatant.ResultsThe culture supernatant from rabbit‐specific strain RDEC‐1, but not from human‐specific enteropathogenic Escherichia coli strain E2348/69, induced an increase in potential difference and short‐circuit current in rabbit ileum mounted in Ussing chambers. This electrical signal was related to chloride ion secretion, was absent in colonic tissue, and was retained in the 30 to 100‐KDa fraction of the supernatant. Preliminary experiments failed to show an involvement of calcium or cyclic nucleotides as intracellular messengers. RDEC‐1 cured of a 42‐MDa plasmid lost the enterotoxicity whereas conjugation of the plasmid into the negative E. coli recipient HB101 resulted in the expression of toxicity.ConclusionsThe authors describe a novel, species‐specific factor that helps to explain RDEC‐1 diarrhea, which may be relevant to the pathogenesis of enteropathogenic Escherichia coli infection.

The Use of an Isolated Earthworm Crop-Gizzard Preparation as a Model for Teaching Smooth Muscle Physiology

Smooth visceral muscle is one of the major types of muscle tissue that students examine in physiology courses. It is an integral part of many organ systems in animals and has several unique characteristics that distinguish it from skeletal and cardiac muscle. It lacks striation, contracts spontaneously, maintains its contractility when stretched, and is controlled by neurotransmitters and hormones. In the classical laboratory for demonstrating smooth muscle properties, part of a mammalian digestive tract like the rabbit ileum or rat jejunum is generally used (Tharp 1997). However, when these and other mammalian smooth muscle tissues are used, they must be handled carefully since contamination will prevent them from contracting. Also, they must be kept at the body temperature of the animal, which is well above room temperature, thus requiring a heated tissue chamber. Other disadvantages include that these animals are costly, require special care, may need the approval of the college or university committee that oversees animal care and usage, and may be unsuitable for use in high school settings. We have developed a comparable laboratory exercise showing the properties of smooth muscle with the crop and gizzard of the earthworm. In this experiment the student removes the combined crop-gizzard from the animal and places it in a tissue bath at room temperature. Once the tissues are connected to a force transducer,

Identification of binding proteins for cholesterol absorption inhibitors as components of the intestinal cholesterol transporter

To identify protein components of the intestinal cholesterol transporter, rabbit small intestinal brush border membrane vesicles were submitted to photoaffinity labeling using photoreactive derivatives of 2-azetidinone cholesterol absorption inhibitors. An integral membrane protein of M r 145.3±7.5 kDa was specifically labeled in brush border membrane vesicles from rabbit jejunum and ileum. Its labeling was concentration-dependently inhibited by the presence of cholesterol absorption inhibitors whereas bile acids, D-glucose, fatty acids or cephalexin had no effect. The inhibitory potency of 2-azetidinones to inhibit photolabeling of the 145 kDa protein correlated with their in vivo activity to inhibit intestinal cholesterol absorption. These results suggest that an integral membrane protein of M r 145 kDa is (a component of) the cholesterol absorption system in the brush border membrane of small intestinal enterocytes.

Invasiveness of Salmonella serotypes Typhimurium and Enteritidis of human gastro-enteritic origin for rabbit ileum: role of LPS, plasmids and host factors.

An organ culture system involving explants of distal rabbit ileum was used to study the roles of lipopolysaccharide (LPS) and plasmids in primary invasiveness for enterocytes in situ of strains of Salmonella serotypes Typhimurium and Enteritidis. Long-chain LPS per se does not confer invasiveness on Typhimurium, as known avirulent, hypo-invasive strains express smooth LPS. However, the invasiveness of a naturally occurring rough isogenic derivative of Salmonella serotype Enteritidis PT 4 was about half that of its wild-type parent. Therefore, smooth LPS appears to play a secondary role in maximising invasiveness. No evidence was found to correlate primary invasiveness for gut of 18 strains of Typhimurium with plasmid profiles in general or with the 60-MDa serovar-specific virulence plasmid in particular. Evidence is presented that strongly suggests a seasonal variability in susceptibility of rabbit gut to invasion by Typhimurium. Although no explanation is given for this summer insusceptibility, the data indicate the importance of the physiological status of the host in relation to susceptibility to invasion by Salmonella.

Contribution of plasmid-encoded fimbriae and intimin to capacity of rabbit-specific enteropathogenic Escherichia coli to attach to and colonize rabbit intestine.

Attachment to the intestinal mucosa is an essential step in the pathogenesis of diarrhea caused by enteropathogenic Escherichia coli (EPEC). Fimbriae and intimin, the outer membrane protein product of the chromosomal eae gene, contribute to this process, but their relative roles and the nature of their interaction are not known. The aim of this study was to determine the relative contribution of plasmid-encoded fimbriae, termed Ral, and intimin to the capacity of rabbit-specific EPEC (REPEC) to attach to the intestinal mucosa of rabbits. To achieve this, we constructed a series of mutants in REPEC strain 83/39 (O15:H-), in which the ralE and eae genes were insertionally inactivated. These strains were then inoculated into ligated loops of rabbit ileum, which were resected 18 h later and examined by light and electron microscopy. The results showed that intimin, but not Ral, is essential for the elicitation of attaching-effacing lesions by REPEC. Nevertheless, a delta eae Ral-bearing mutant adhered to the intestinal epithelium to the same extent as its eae-positive parent and far more extensively than an eae(+) delta ral strain. To examine the contribution of Ral and intimin to colonization of rabbit intestine, we fed these strains to weanling rabbits, which were killed 4 days later, so that the number of bacteria in various regions of the intestine could be determined. The results indicated that strain 83/39 requires both Ral and intimin to colonize the intestine successfully and that a delta eae delta ralE double mutant was incapable of colonizing the intestine. Taken together, these findings indicate that Ral and intimin act independently as adhesion factors of REPEC strain 83/39 and that this strain carries no other significant colonization factor. When both Ral and intimin are present, they appear to act cooperatively, with Ral-mediated adhesion preceding that mediated by intimin.

Absence of intestinal secretion on supernatants from macrophages stimulated with Clostridium difficile toxin B on rabbit ileum

Several studies have documented the involvement of both Clostridium difficile, toxins, A and B in the pathogenesis of antibiotic-associated diarrhea. Recently, we demonstrated that IL-1β is the intestinal secretory factor released by macrophages stimulated with toxin A. The aim of this study was to evaluate the importance of macrophages stimulated with toxin B on rabbit ileal ion transport. The changes in ion transport were analyzed by studying the short-circuit current of the rabbit ileal mucosa mounted in Üssing chambers. The supernatants of macrophages treated with toxin B (3.6×10 −7 M) had no effect on the ion transport (change in short-circuit current ΔI sc =28.0±9.2 vs. control=26.8±3.6 μA cm −2). Supernatants of macrophages stimulated with toxin A (3.2×10 −7 M), our positive control, induced a significant change in ileal ion transport (Δ I sc=55.2±5.7 mA cm −2). It was also observed that, like toxin A, toxin B stimulated macrophages to produce TNF-α (555.0±37.9 pg/ml vs. control=182.0±39.8 pg/ml; p<0.05). Nevertheless, in contrast to toxin A, toxin B did not stimulate IL-1β synthesis (28.0±7.5 pg/ml vs. control=40.0±14.4 pg/ml; p>0.05). We conclude that the supernatants of macrophages stimulated with toxin B are not able to stimulate ion transport and that both toxins stimulate the genesis of TNF-α, but only toxin A induces the synthesis of IL-1β, which, we have earlier reported, causes an electrogenic intestinal response in rabbit ileum.

Vibrio cholerae ACE stimulates Ca(2+)-dependent Cl(-)/HCO(3)(-) secretion in T84 cells in vitro.

ACE, accessory cholera enterotoxin, the third enterotoxin in Vibrio cholerae, has been reported to increase short-circuit current (I(sc)) in rabbit ileum and to cause fluid secretion in ligated rabbit ileal loops. We studied the ACE-induced change in I(sc) and potential difference (PD) in T84 monolayers mounted in modified Ussing chambers, an in vitro model of a Cl(-) secretory cell. ACE added to the apical surface alone stimulated a rapid increase in I(sc) and PD that was concentration dependent and immediately reversed when the toxin was removed. Ion replacement studies established that the current was dependent on Cl(-) and HCO(3)(-). ACE acted synergistically with the Ca(2+)-dependent acetylcholine analog, carbachol, to stimulate secretion in T84 monolayers. In contrast, the secretory response to cAMP or cGMP agonists was not enhanced by ACE. The ACE-stimulated secretion was dependent on extracellular and intracellular Ca(2+) but was not associated with an increase in intracellular cyclic nucleotides. We conclude that the mechanism of secretion by ACE involves Ca(2+) as a second messenger and that this toxin stimulates a novel Ca(2+)-dependent synergy.

Supernatants from Macrophages Stimulated with Microcystin‐LR Induce Electrogenic Intestinal Response in Rabbit Ileum

Microcystin-LR is a cyclic heptapeptide hepatotoxin produced by the cyanobacterium Microcystis aeruginosa. This microorganism often forms toxic blooms in freshwater lakes and reservoirs for drinking water supply, producing serious disorders in humans and animals. Some have suggested that certain biological activities of microcystin may depend upon the stimulation of immune cells. Therefore, the aims of this research were to examine electrogenic intestinal secretion, in vitro, caused by the supernatants from macrophages stimulated with microcystin-LR, as well as to investigate the presence of interleukin-1beta and tumour necrosis factor-alpha in these supernatants. We found that the supernatants of macrophages stimulated with microcystin-LR (0.1, 0.3 and 1.0 microg/ml) caused electrogenic intestinal effects (change in short circuit currents (delta SCC)=57.6, 50.8 and 73.3, respectively, versus control=19.6 microA.cm(-2)) in a time-dependent way (microcystin-LR (1.0 microg/ml)=63.2, 108.8, 120.4 and 132.3 microA.cm(-2) at time 0, 40, 50 and 60 min., respectively). In addition, the intestinal secretory activity present in these supernatants was blocked (57%) by the prior treatment of macrophages with dexamethasone. We also demonstrated that microcystin-LR (0.1, 0.3 and 1.0 ,microg/ml) is capable of stimulating the synthesis of tumour necrosis factor-alpha (375.4, 369.0 and 610.8 pg/ml, respectively, versus control=165.0 pg/ml) and interleukin-1beta (198.9, 189.3 and 522.1 pg/ml, respectively, versus control=39.7 pg/ml). These findings demonstrate that microcystin-LR induces the release of interleukin-1beta and tumour necrosis factor-alpha by peritoneal macrophages in vitro, and that the supernatants from these macrophages induce electrogenic secretion in rabbit ileal mucosa.

Nphe 1]nociceptin-(1–13)NH 2 selectively antagonizes nociceptin effects in the rabbit isolated ileum

When suspended in vitro in isolated organ baths, segments of the rabbit ileum show a fairly strong and stable spontaneous activity, which derives from the continuous release of acetylcholine and the activation of muscarinic receptors, since the activity is completely eliminated by atropine. Dynorphin A (pEC 50: 8.6±0.07), neuropeptide Y and its congener human pancreatic polypeptide (pEC 50: 9.40±0.10), and nociceptin (pEC 50: 8.08±0.12) dose-dependently inhibit the spontaneous activity through the activation of receptors, which are specifically antagonised respectively by naloxone (p A 2: 7.17±0.12), 2-(naphtalen-1-ylamino)-3-phenylpropionitrile (JCF 104; p A 2: 5.80±0.10), and [Nphe 1]nociceptin-(1–13)NH 2 (p A 2: 6.17±0.19). This last compound, a selective nociceptin-receptor (OP 4) antagonist, inhibits the effect of nociceptin in a competitive manner, as demonstrated by Schild analysis. [Nphe 1]nociceptin-(1–13)NH 2 also antagonizes the effects of other OP 4 receptor ligands such as the full agonist, nociceptin-(1–13)-NH 2, and the partial agonists, [Phe 1ψ(CH 2–NH)Gly 2]nociceptin-(1–13)-NH 2 (intrinsic activity ( α E)=0.5) and Ac-RYYWK-NH 2 ( α E=0.5), with p A 2 values ranged from 5.8 to 6.2. These results indicate that the functional site mediating the inhibitory effect of nociceptin in the rabbit ileum, is pharmacologically identical to the OP 4 sites of other species (mouse, rat, guinea pig, man), since the potencies (p A 2 values) of the pure and competitive antagonist [Nphe 1]nociceptin-(1–13)NH 2 is very similar to the values obtained in the other species. Moreover, the rabbit ileum is one of the few isolated organs that allow classifying compounds, which interact with OP 4 receptors as full agonists, partial agonists, or pure antagonists.

Effect of cholera toxin on glutamine metabolism and transport in rabbit ileum.

The aim of the present study was to evaluate the effect of cholera toxin on energy balance from intestinal glutamine metabolism and oxidation, glutamine-dependent sodium absorption, and cholera toxin-dependent ion flux. Cholera toxin-stimulated sodium and L-glutamine ileal transport and metabolism were studied in Ussing chambers. Glutamine (10 mM) transport and metabolism were simultaneously studied using (14)C flux and HPLC. In the same tissues, the flux of each amino acid was studied by HPLC, and glutamine metabolism and oxidation were studied by the determination of amino acid specific activity and (14)CO(2) production. In control tissues, glutamine stimulated sodium absorption and was mainly oxidized. The transepithelial flux of intact glutamine represented 45% of glutamine flux across the luminal membrane. The other metabolites were glutamate and, to a lesser degree, citrulline, ornithine, and proline. Cholera toxin did not alter glutamine-stimulated sodium absorption, glutamine oxidation, transport, and metabolism. In conclusion, the present results indicate that cholera toxin does not alter glutamine intestinal function and metabolism. In addition, approximately 95% of the energy provided by glutamine oxidation remains available to the enterocyte.

Factors limiting the oral bioavailability of N-acetylglucosaminyl- N-acetylmuramyl dipeptide (GMDP) and enhancement of absorption in rats by delivery in a water-in-oil microemulsion

The bioavailability (BA) of radio-labelled N-acetylglucosaminyl- N-acetylmuramyl dipeptide (GMDP) was low when administered by oral gavage as an aqueous solution to conscious male Sprague–Dawley rats (8.3±4.4% (mean±S.D., n=3)). To assess the likely factors contributing to the poor BA of GMDP, the stability of GMDP in the lumen of the gastrointestinal (GI) tract was examined in vitro, using ex vivo GI contents. GMDP was degraded by the contents of the small intestine, caecum and large intestine but was more stable in stomach contents. The permeability coefficient (p app) of GMDP in isolated sections of rabbit ileum was 1.67×10 −6 cm/s in the mucosal to serosal direction and was not significantly different in the serosal to mucosal direction, indicating that GMDP is poorly permeable and passively transported across the intestinal wall. First pass metabolism was considered to be unlikely to be the primary limitation to the oral bioavailability of GMDP and therefore, that the oral bioavailability of GMDP was likely limited by instability in the lumen of the gastrointestinal tract and low intestinal permeability. A water-in-oil (w/o) microemulsion formulation subsequently developed to address these problems was trialed in a preliminary bioavailability study in rats and enhanced the bioavailability of GMDP ten-fold when administered intraduodenally, indicating that w/o microemulsions may represent a viable mechanism for enhancing the bioavailability of poorly GI-stable and poorly permeable peptide-based molecules.

Effect of chronic inflammation on ileal short-chain fatty acid/bicarbonate exchange.

Short-chain fatty acids (SCFA) have been demonstrated to at least partially ameliorate chronic intestinal inflammation. However, whether and how intestinal SCFA absorption may be altered during chronic intestinal inflammation is unknown. A rabbit model of chronic ileitis produced by coccidia was used to determine the effect of chronic inflammation on ileal SCFA/HCO(-)(3) exchange. SCFA/HCO(-)(3) exchange was present in the brush-border membrane (BBM) of villus but not crypt cells from normal rabbit ileum. An anion-exchange inhibitor, DIDS, significantly inhibited SCFA/HCO(-)(3) exchange. Extravesicular Cl(-) did not alter the uptake of SCFA, suggesting that SCFA/HCO(-)(3) exchange is a transport process distinct from Cl(-)/HCO(-)(3) exchange. In chronically inflamed ileum, SCFA/HCO(-)(3) exchange was also present only in BBM of villus cells. The exchanger was sensitive to DIDS and was unaffected by extravesicular Cl(-). However, SCFA/HCO(-)(3) exchange was significantly reduced in villus cell BBM vesicles (BBMV) from chronically inflamed ileum. Kinetic studies demonstrated that the maximal rate of uptake of SCFA, but not the affinity for SCFA, was reduced in chronically inflamed rabbit ileum. These data demonstrate that a distinct SCFA/HCO(-)(3) exchange is present on BBMV of villus but not crypt cells in normal rabbit ileum. SCFA/HCO(-)(3) exchange is inhibited in chronically inflamed rabbit ileum. The mechanism of inhibition is most likely secondary to a reduction in transporter numbers rather than altered affinity for SCFA.