1 Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Dr Molewaterplein 40, 3015 GD Rotterdam, The Netherlands
2 Department of Comparative Medicine, Stanford University School of Medicine, Stanford, CA, USA
Correspondence
Arnoud H.M. van Vliet
a.h.m.vanvliet{at}erasmusmc.nl
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
H. hepaticus expresses a nickel co-factored urease enzyme (Beckwith et al., 2001). Urease is a multimeric, nickel-containing enzyme produced by many pathogenic and non-pathogenic bacteria (Burne & Chen, 2000
). In Helicobacter species, the urease enzyme consists of UreA and UreB subunits (Beckwith et al., 2001
). These are encoded by the ureA and ureB genes and are located in an operon, also containing the ureI gene (encoding a urea channel), and the ureEFGH genes encoding accessory proteins involved in enzyme activation (Beckwith et al., 2001
; Burne & Chen, 2000
; Skouloubris et al., 1998
). Active urease converts urea into ammonia and bicarbonate; the ammonia produced is thought to mediate protection against acidic microenvironments, but may also serve as a nitrogen source (Williams et al., 1996
).
In gastric Helicobacter species like H. pylori and Helicobacter mustelae, urease is an important virulence factor (Andrutis et al., 1995; Tsuda et al., 1994
). The ammonia produced is thought to allow protection against acidic environments, and mutants lacking urease are unable to colonize the gastric environments in different animal models (Andrutis et al., 1995
; Tsuda et al., 1994
). In H. pylori, urease expression and enzyme activity are controlled by an intricate regulatory network, centred on the environmental pH and the availability of the cofactor nickel (Scott et al., 2002
; van Vliet et al., 2004b
). For H. hepaticus and other urease-positive enterohepatic Helicobacter species, the role of urease in metabolism or pathogenesis has not yet been established.
Recently, differences between the urease and nickel-transport genes of H. hepaticus and H. pylori were reported (Beckwith et al., 2001; Suerbaum et al., 2003
), which suggested that there may be differences in regulation of urease expression and activity between these two Helicobacter species. Here it is demonstrated that urease activity in H. hepaticus is nickel-responsive, but solely at the post-translational level. In contrast to H. pylori, H. hepaticus urease activity is not induced at acidic pH, and H. hepaticus does not grow or survive at pH 3·0. This suggests that the urease system is not a universal acid-resistance factor of ureolytic Helicobacter species, and that its functions may differ between gastric and enterohepatic Helicobacter species.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acid shock survival.
H. hepaticus was grown for 24 h in BBC at pH 7·0 at a starting OD600 of 0·05. Bacteria were then harvested by centrifugation for 10 min at 4000 g and resuspended to a final OD600 of 0·2 in PBS adjusted to pH 7·0, 5·5 or 3·0. When indicated, PBS was supplemented with NiCl2 or urea to a final concentration of 100 µM and 0·5 mM, respectively. Cells were incubated for 30 min at 37 °C in microaerobic conditions. Subsequently 5 µl of tenfold dilutions were spotted on Dent agar. Plates were incubated for 2 days at 37 °C in microaerobic conditions and the presence or absence of growth at the spots was assessed by visual inspection (Baillon et al., 1999). Differences in the highest dilution still containing growth between different conditions indicated differences in survival rates.
Urease enzyme assay.
Urease activity was determined in freshly sonicated lysates by measuring ammonia production from hydrolysis of urea, as described previously (van Vliet et al., 2001). The concentration of ammonia in the samples was inferred from a standard NH4Cl concentration curve. Enzyme activity was expressed as µmol urea substrate hydrolysed min1 (mg protein)1.
RNA analysis.
RNA was isolated from H. hepaticus using Trizol reagent (Invitrogen), according to the manufacturer's instructions. Gel electrophoresis of RNA, transfer to positively charged nylon membranes (Roche), cross-linking, hybridization to DIG-labelled specific RNA probes and detection of bound probe were performed as described previously (van Vliet et al., 2001). Probes specific for H. hepaticus ureA and ureB were synthesized by in vitro transcription using T7 RNA polymerase (Roche) and PCR products obtained with primers HhUreA-F1 (5'-TGCATTATGCTGGGGCACTA-3') and HhUreA-R1-T7 (5'-ctaatacgactcactatagggagaATAGGTCTATCGCCCTTATG-3'), and HhUreB-F1 (5'-TGGTAAAGCGGGAAATCCAG-3') and HhUreB-R1 T7 (5'-ctaatacgactcactatagggagaGTGTGCAGGTAGTAGCGTTTG-3'). Lower-case letters indicate the T7 promoter used for in vitro transcription.
Construction of an H. hepaticus ureB mutant.
The ureB gene of H. hepaticus ATCC 51449 was amplified using primers HhUreB-mutF1 (5'-TCCTGTGCCTCCACCAAT-3') and HhUreB-mutR1 (5'-GCATTATGCTGGGGCACT-3'), and cloned in pGEM-Teasy (Promega), resulting in plasmid pJS10. The ureB gene was subsequently interrupted by insertion of the chloramphenicol-resistance gene from pAV35 (van Vliet et al., 1998) in the unique BsmI site, resulting in plasmid pJS11. This plasmid was introduced into E. coli ER1793 and subsequently used for natural transformation of H. hepaticus ATCC 51449. Chloramphenicol-resistant colonies isolated were designated 51449ureB. Two colonies derived from independent transformations were tested, and both colonies gave identical results in all experiments. Correct allelic replacement of the wild-type ureB gene with the interrupted version was confirmed by PCR.
Protein analysis.
H. hepaticus wild-type and ureB mutant cells were grown for 24 h in unsupplemented BBC medium or BBC medium supplemented with 100 µM NiCl2, centrifuged at 4000 g for 10 min at room temperature and resuspended in PBS pH 7·4 to a final OD600 of 10. The cells were subsequently lysed by sonication for 15 s on ice, using an MSE Soniprep 150 at amplitude 6. The protein concentration of lysates was determined using the bicinchoninic acid method (Pierce) using bovine serum albumin as standard. Proteins were separated by SDS-PAGE on a 10 % (w/v) polyacrylamide gel and stained with Coomassie brilliant blue. Western blotting was performed by electrotransfer of proteins onto nitrocellulose membrane (Roche). The blot was probed with antibodies raised in rabbits to Helicobacter felis UreA or UreB (Intervet International). Bound antibodies were visualized with swine anti-rabbit antibodies labelled with alkaline phosphatase (Promega), using 5-bromo-4-chloro-3-indolyl phosphate/nitro-blue tetrazolium (BCIP/ NBT) (Promega) as substrate.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
H. hepaticus urease activity is acid-independent, but nickel-induced
To determine the effect of medium pH on growth and urease activity, H. hepaticus was grown for 24 h in BBC medium adjusted to pH 5·5, 6·0 and 7·0. These pH values were selected on the basis of their use in previous studies with H. pylori, as pH 5·5 and 6·0 are thought to resemble the pH of the mucus layer (van Vliet et al., 2004b). pH 5·5 was the lowest pH value allowing growth of H. hepaticus (Fig. 1
). The pH of the medium did not change significantly during 24 h growth. Urease activity in H. hepaticus was not significantly affected by the pH of the medium (Fig. 2
a), while urease activity in the positive control H. pylori increased at pH 6·0 and pH 5·5 (Fig. 2b
), consistent with earlier data (Scott et al., 2002
; van Vliet et al., 2004b
).
|
Nickel-responsive induction of urease activity in H. hepaticus is mediated at the post-translational level
To identify at which level the nickel-responsive induction of urease activity in H. hepaticus was mediated, the effect of nickel on transcription of H. hepaticus urease genes and expression of urease protein was determined (Fig. 3). Nickel supplementation of BBC medium did not affect transcription of either the ureA or ureB genes (Fig. 3a
). Likewise, expression of the UreA and UreB proteins was not altered by nickel supplementation, as was shown for both UreA and UreB by Western immunoblotting using antibodies to H. felis UreA and UreB protein (Fig. 3b
). The Western blot data were independently confirmed using an isogenic ureB mutant of H. hepaticus ATCC 51449 (Fig. 3c
). This ureB mutant showed an absence of UreB expression, and did not have any detectable urease activity; it did not show any significant changes in growth under the tested conditions (data not shown).
|
Nickel-responsive induction of urease activity in H. hepaticus is growth-phase-independent
The effect of growth phase on nickel-responsive induction of urease was determined for H. hepaticus and H. pylori (Fig. 4). Nickel supplementation did not significantly affect the growth of H. hepaticus or H. pylori, and both species reached late-exponential phase after 24 h (Fig. 4a
). H. hepaticus already reached maximum urease activity at approximately 12 h of growth in nickel-supplemented medium (Fig. 4b
). In contrast, urease activity in H. pylori increased steadily in nickel-supplemented cultures, and reached its maximum at approximately 18 h of growth, consistent with the requirement for transcription and translation of extra urease protein for increased urease activity during this period of time (Fig. 4b
). This indicates that in H. hepaticus urease activity peaks during early exponential phase, further supporting the hypothesis that nickel induction of urease activity is mediated by enzyme activation rather than increased expression of urease enzyme.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study it has been demonstrated that H. hepaticus is acid-sensitive, and that addition of urea does not increase its survival at pH 3·0 (Fig. S1). In contrast, addition of urea does allow gastric Helicobacter species like H. pylori to survive such acidic conditions. Taken together, these findings suggest that the urease enzyme of H. hepaticus is not involved in acid resistance. Consistent with this was the lack of acid-responsive induction of urease activity in H. hepaticus (Fig. 2a). However, urease activity in H. hepaticus is nickel-responsive, but this regulation is mediated solely at the post-translational level, probably through the activation of preformed apo-enzyme (Figs 3 and 4
). During revision of this paper, nickel-responsive induction of H. hepaticus urease activity was confirmed in an independent study (Mehta et al., 2005
), indicating that the observed nickel induction of H. hepaticus urease activity is not due to the conditions employed in our study.
Despite overall similarities of the H. hepaticus and H. pylori urease gene clusters, there are notable differences. Both urease gene clusters consist of two structural genes (ureAB) and five accessory genes (ureIEFGH) (Beckwith et al., 2001), but the ureBureI intergenic distance of H. hepaticus is much shorter (9 bp) than that of H. pylori (200 bp). This indicates that in H. hepaticus there is probably no promoter directly upstream of the ureI gene, thus limiting the possibilities for transcriptional and post-transcriptional regulation (Akada et al., 2000
; van Vliet et al., 2001
). Furthermore, the overall amino acid sequence of UreI in both species is well conserved, but several amino acid residues which were shown to be required for acid activation of the H. pylori UreI urea channel are absent in the H. hepaticus UreI protein (Weeks et al., 2004
). This is consistent with the extragastric lifestyle of H. hepaticus and its inability to grow or survive at pH 3·0 (Figs 1
and S1). Whether this phenotype is due to the lack of acid-responsive regulation of urease activity, absence of a ureI promoter or absence of the acid activation of the UreI urea channel remains to be established.
Enzymic degradation of urea by urease results in the production of ammonia and bicarbonate, and both reaction products are thought to play an important part in bacterial pathogenesis. Ammonia serves as nitrogen source and may function in acid resistance (Burne & Chen, 2000; Scott et al., 2002
; Williams et al., 1996
), whereas bicarbonate may also function in acid resistance as well as in modulating the immune system of the host (Kuwahara et al., 2000
; Marcus et al., 2005
). Expression of urease in bacteria is controlled by different stimuli, such as urea availability, environmental pH, nitrogen status of the cell, or growth phase (Burne & Chen, 2000
).
In this study it has been demonstrated that urease activity in H. hepaticus is nickel-responsive. This form of urease regulation has to date only been identified for two other urease systems, of H. pylori (van Vliet et al., 2001) and Streptococcus salivarius (Chen & Burne, 2003
). The enzymic activity of H. hepaticus urease is induced by nickel supplementation of the growth medium, but this induction is mediated solely at the post-translational level (Fig. 3
), probably by activation of urease apo-enzyme, as was previously described for S. salivarius (Chen & Burne, 2003
). In contrast, the H. pylori urease system is nickel-induced at the enzyme activity and transcriptional level (van Vliet et al., 2001
). The nickel- and acid-responsive transcriptional regulation of urease observed in H. pylori may be a specific adaptation to the gastric lifestyle. This suggests that the proposed link in H. pylori between intracellular nickel availability and environmental pH (van Vliet et al., 2004a
) may not be universal in the genus Helicobacter.
In H. pylori, regulation of urease activity is dependent on nickel and the NikR regulatory protein (van Vliet et al., 2002). Mutants lacking the NikR regulators are unable to colonize the murine gastric mucosa (Bury-Mone et al., 2004
). A NikR homologue is also present in H. hepaticus (Suerbaum et al., 2003
), and it can be envisaged that NikR may indirectly affect urease activity via regulation of nickel uptake. In H. pylori, NikR regulates NixA-mediated nickel uptake (Ernst et al., 2005
), and while the H. hepaticus genome sequence does not contain a nixA orthologue (Suerbaum et al., 2003
), it does contain a putative nickel-uptake ABC transporter located adjacent to the urease operon (Beckwith et al., 2001
). However, up to now the H. hepaticus nikR gene has proven refractory to insertional mutagenesis (C. Belzer & A. H. M. van Vliet, unpublished results), and thus a role of NikR in regulation of urease expression of H. hepaticus cannot be established yet.
In conclusion, H. hepaticus is acid-sensitive and lacks regulatory mechanisms mediating acid- and nickel-responsive regulation of urease expression. These characteristics may reflect its extragastric niche, and may be directly linked with the animal host colonized and/or the respective target organ. We hypothesize that enterohepatic Helicobacter species do not require high levels of urease activity in the rodent gut. The high levels of urease activity observed in gastric Helicobacter species (Bury-Mone et al., 2003; Scott et al., 2000
) are likely to be an adaptation that allows them to thrive in the gastric environment, albeit at a high metabolic cost. Regulation of urease expression and activity will allow them to adapt to changes in acidity observed during fasting or feeding. In contrast, urease-positive enterohepatic Helicobacter species might use their urease system for nitrogen metabolism, securing a constant supply of ammonia. The diversity in urease function and regulatory responses in Helicobacter species is a prime example of the adaptation required for chronic colonization of host tissues.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andrutis, K. A., Fox, J. G., Schauer, D. B., Marini, R. P., Murphy, J. C., Yan, L. & Solnick, J. V. (1995). Inability of an isogenic urease-negative mutant stain of Helicobacter mustelae to colonize the ferret stomach. Infect Immun 63, 37223725.[Abstract]
Baillon, M. L. A., van Vliet, A. H. M., Ketley, J. M., Constantinidou, C. & Penn, C. W. (1999). An iron-regulated alkyl hydroperoxide reductase (AhpC) confers aerotolerance and oxidative stress resistance to the microaerophilic pathogen Campylobacter jejuni. J Bacteriol 181, 47984804.
Beckwith, C. S., McGee, D. J., Mobley, H. L. & Riley, L. K. (2001). Cloning, expression, and catalytic activity of Helicobacter hepaticus urease. Infect Immun 69, 59145920.
Blaser, M. J. & Atherton, J. C. (2004). Helicobacter pylori persistence: biology and disease. J Clin Invest 113, 321333.
Burne, R. A. & Chen, Y. Y. (2000). Bacterial ureases in infectious diseases. Microbes Infect 2, 533542.[CrossRef][Medline]
Bury-Mone, S., Skouloubris, S., Dauga, C., Thiberge, J. M., Dailidiene, D., Berg, D. E., Labigne, A. & De Reuse, H. (2003). Presence of active aliphatic amidases in Helicobacter species able to colonize the stomach. Infect Immun 71, 56135622.
Bury-Mone, S., Thiberge, J. M., Contreras, M., Maitournam, A., Labigne, A. & De Reuse, H. (2004). Responsiveness to acidity via metal ion regulators mediates virulence in the gastric pathogen Helicobacter pylori. Mol Microbiol 53, 623638.[CrossRef][Medline]
Chen, Y. Y. & Burne, R. A. (2003). Identification and characterization of the nickel uptake system for urease biogenesis in Streptococcus salivarius 57.I. J Bacteriol 185, 67736779.
Ernst, F. D., Kuipers, E. J., Heijens, A., Sarwari, R., Stoof, J., Penn, C. W., Kusters, J. G. & van Vliet, A. H. M. (2005). The nickel-responsive regulator NikR controls activation and repression of gene transcription in Helicobacter pylori. Infect Immun 73, 72527258.
Fox, J. G., Dewhirst, F. E., Tully, J. G., Paster, B. J., Yan, L., Taylor, N. S., Collins, M. J., Jr, Gorelick, P. L. & Ward, J. M. (1994). Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice. J Clin Microbiol 32, 12381245.[Abstract]
Franklin, C. L., Riley, L. K., Livingston, R. S., Beckwith, C. S., Besch-Williford, C. L. & Hook, R. R., Jr (1998). Enterohepatic lesions in SCID mice infected with Helicobacter bilis. Lab Anim Sci 48, 334339.[Medline]
Franklin, C. L., Riley, L. K., Livingston, R. S., Beckwith, C. S., Hook, R. R., Jr, Besch-Williford, C. L., Hunziker, R. & Gorelick, P. L. (1999). Enteric lesions in SCID mice infected with "Helicobacter typhlonicus", a novel urease-negative Helicobacter species. Lab Anim Sci 49, 496505.[Medline]
Kusters, J. G., Gerrits, M. M., Van Strijp, J. A. & Vandenbroucke-Grauls, C. M. J. E. (1997). Coccoid forms of Helicobacter pylori are the morphologic manifestation of cell death. Infect Immun 65, 36723679.[Abstract]
Kuwahara, H., Miyamoto, Y., Akaike, T., Kubota, T., Sawa, T., Okamoto, S. & Maeda, H. (2000). Helicobacter pylori urease suppresses bactericidal activity of peroxynitrite via carbon dioxide production. Infect Immun 68, 43784383.
Lee, A., Chen, M., Coltro, N., O'Rourke, J., Hazell, S., Hu, P. & Li, Y. (1993). Long term infection of the gastric mucosa with Helicobacter species does induce atrophic gastritis in an animal model of Helicobacter pylori infection. Zentralbl Bakteriol 280, 3850.[Medline]
Marcus, E. A., Moshfegh, A. P., Sachs, G. & Scott, D. R. (2005). The periplasmic alpha-carbonic anhydrase activity of Helicobacter pylori is essential for acid acclimation. J Bacteriol 187, 729738.
Maurer, K. J., Ihrig, M. M., Rogers, A. B., Ng, V., Bouchard, G., Leonard, M. R., Carey, M. C. & Fox, J. G. (2005). Identification of cholelithogenic enterohepatic Helicobacter species and their role in murine cholesterol gallstone formation. Gastroenterology 128, 10231033.[CrossRef][Medline]
Mehta, N. S., Benoit, S., Mysore, J. V., Sousa, R. S. & Maier, R. J. (2005). Helicobacter hepaticus hydrogenase mutants are deficient in hydrogen-supported amino acid uptake and in causing liver lesions in A/J mice. Infect Immun 73, 53115318.
Nilsson, H. O., Castedal, M., Olsson, R. & Wadstrom, T. (1999). Detection of Helicobacter in the liver of patients with chronic cholestatic liver diseases. J Physiol Pharmacol 50, 875882.[Medline]
Queiroz, D. M. & Santos, A. (2001). Isolation of a Helicobacter strain from the human liver. Gastroenterology 121, 10231024.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Scott, D. R., Marcus, E. A., Weeks, D. L., Lee, A., Melchers, K. & Sachs, G. (2000). Expression of the Helicobacter pylori ureI gene is required for acidic pH activation of cytoplasmic urease. Infect Immun 68, 470477.
Scott, D. R., Marcus, E. A., Weeks, D. L. & Sachs, G. (2002). Mechanisms of acid resistance due to the urease system of Helicobacter pylori. Gastroenterology 123, 187195.[CrossRef][Medline]
Skouloubris, S., Thiberge, J. M., Labigne, A. & De Reuse, H. (1998). The Helicobacter pylori UreI protein is not involved in urease activity but is essential for bacterial survival in vivo. Infect Immun 66, 45174521.
Solnick, J. V. & Schauer, D. B. (2001). Emergence of diverse Helicobacter species in the pathogenesis of gastric and enterohepatic diseases. Clin Microbiol Rev 14, 5997.
Suerbaum, S., Josenhans, C., Sterzenbach, T. & 19 other authors (2003). The complete genome sequence of the carcinogenic bacterium Helicobacter hepaticus. Proc Natl Acad Sci U S A 100, 79017906.
Tomb, J. F., White, O., Kerlavage, A. R. & 39 other authors (1997). The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388, 539547.[CrossRef][Medline]
Tsuda, M., Karita, M., Morshed, M. G., Okita, K. & Nakazawa, T. (1994). A urease-negative mutant of Helicobacter pylori constructed by allelic exchange mutagenesis lacks the ability to colonize the nude mouse stomach. Infect Immun 62, 35863589.[Abstract]
van Vliet, A. H. M., Wooldridge, K. G. & Ketley, J. M. (1998). Iron-responsive gene regulation in a Campylobacter jejuni fur mutant. J Bacteriol 180, 52915298.
van Vliet, A. H. M., Kuipers, E. J., Waidner, B. & 7 other authors (2001). Nickel-responsive induction of urease expression in Helicobacter pylori is mediated at the transcriptional level. Infect Immun 69, 48914897.
van Vliet, A. H. M., Poppelaars, S. W., Davies, B. J., Stoof, J., Bereswill, S., Kist, M., Penn, C. W., Kuipers, E. J. & Kusters, J. G. (2002). NikR mediates nickel-responsive transcriptional induction of urease expression in Helicobacter pylori. Infect Immun 70, 28462852.
van Vliet, A. H. M., Ernst, F. D. & Kusters, J. G. (2004a). NikR-mediated regulation of Helicobacter pylori acid adaptation. Trends Microbiol 12, 489494.[CrossRef][Medline]
van Vliet, A. H. M., Kuipers, E. J., Stoof, J., Poppelaars, S. W. & Kusters, J. G. (2004b). Acid-responsive gene induction of ammonia-producing enzymes in Helicobacter pylori is mediated via a metal-responsive repressor cascade. Infect Immun 72, 766773.
Verhoef, C., Pot, R. G. J., de Man, R. A., Zondervan, P. E., Kuipers, E. J., IJzermans, J. N. & Kusters, J. G. (2003). Detection of identical Helicobacter DNA in the stomach and in the non-cirrhotic liver of patients with hepatocellular carcinoma. Eur J Gastroenterol Hepatol 15, 11711174.[CrossRef][Medline]
Ward, J. M., Fox, J. G., Anver, M. R. & 7 other authors (1994). Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter species. J Natl Cancer Inst 86, 12221227.[Abstract]
Weeks, D. L., Gushansky, G., Scott, D. R. & Sachs, G. (2004). Mechanism of proton gating of a urea channel. J Biol Chem 279, 99449950.
Williams, C. L., Preston, T., Hossack, M., Slater, C. & McColl, K. E. (1996). Helicobacter pylori utilises urea for amino acid synthesis. FEMS Immunol Med Microbiol 13, 8794.[CrossRef][Medline]
Received 10 May 2005;
revised 6 September 2005;
accepted 19 September 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |